Tesis sobre el tema "Human telomeric"

In humans there are two proteins that specifically bind the duplex telomeric DNA, human TTAGGG Repeat Binding Factor 1 and 2 (hTRF1 and hTRF2). Although similar in sequence and architecture, hTRF1 and hTRF2 play different roles. hTRF1 regulates telomere length while hTRF2 acts to cap telomere ends and prevents them being recognised as DNA breaks. This work has used biochemical and structural techniques to gain insights into three telomeric complexes, hTRF2 and its cofactor hRap1 and hTRF1 and hTRF2 DNA-binding domains in complex with telomeric DNA. The first part of this thesis covers the characterisation of the interaction between hTRF2 and its partner hRap1. Minimal interaction domains of hTRF2 and hRap1 were successfully defined. Constructs were then designed for use in crystallisation trials, but as yet no crystals have been attained. The second part of this thesis involves the interactions of hTRF1 and hTRF2 with telomeric DNA. Biochemical studies were carried out with the DNA-binding domains to compare binding activity. Then, hTRF1 and hTRF2 DNA-binding domains were co-crystallised with telomeric DNA fragments and their structures determined at high resolution. The structures of the two complexes are highly similar, but there are subtle differences in the details of DNA binding. The structures show that much of the sequence specificity of hTRF1 and hTRF2 is conferred by the presence of water molecules at the protein-DNA interface and that specific sequence recognition as well as binding of duplex telomeric DNA is conserved from yeasts to mammals.

ABSTRACT A major cause of genetic disease is associated with chromosomal imbalances, such as deletions (subtelomeric, terminal and interstitial), duplications, and unbalanced translocations present in a particular chromosome segment. The diagnosis of many genetic diseases remains problematic. This is due in part to difficulty in detection of DNA copy number changes, when these are either too small (for conventional cytogenetics) or too large, for standard molecular approaches. From this viewpoint, the development of new screening methods with improvement of resolution is very important. Genome-wide screening at a molecular level began to appear feasible with the completion of the human genome sequence. From this beginning, high-resolution whole-genome technologies could be envisaged, to improve the diagnostic detection rate for even the smallest of chromosomal imbalances. The technique known as ???array-based comparative genomic hybridization??? (array-CGH) does allow such a high-resolution screening, by use of reference DNA probes, printed onto arrays, thus consisting of thousands of genomic clones. In this study we extensively investigated many major aspects of array-CGH technology from preparation of microarray probes and printing microarray slides, to a development of custom protocols and custom softwares for data processing and analysis. We have trailed several array types and protocols, direct and indirect DNA labelling techniques and, as a result, we have achieved the practical application which was our target at the onset of this work. This was to use a modified array-CGH method, as a robust and economical diagnostic test in detection of deletions and duplications within the human genome. The project has been successful, in terms of one very important outcome: The laboratory in which this work was done is now the leading clinical diagnostic lab in this field, in Australia???s most populous state of New South Wales. That achievement would not have been possible without a very lengthy period of developmental work, including that which comprises much of this thesis.

Telomeres are nucleoprotein structures that contain non-coding (TTAGGG) tandem repeats and associated telomere binding proteins at the end of chromosomes. As a consequence of end-replication losses, telomeres undergo gradual erosion with ongoing cell division. It is hypothesised that in addition to the end-replication problem, mutational mechanisms may contribute to telomere erosion by generating large-scale telomeric deletion events. As short dysfunctional telomeres are capable of fusion to other chromosome ends, large-scale telomeric deletions can lead to genomic instability which in turn may drive tumour progression. The primary aim of this thesis was to investigate putative mutational mechanisms that could lead to large-scale telomeric deletion. The role of oxidative stress and it potential contribution to telomere dynamics was assessed. The induction of fragility and replication inhibition at telomeres was also examined. Furthermore, the role that G-quadruplex structure within telomere repeat sequences and the possible induction of replication fork stalling and resolution as single or double stranded breaks was also considered as a mutational mechanism that could lead to telomere deletion. High-resolution analysis of telomere dynamics using Single Telomere Length Analysis (STELA), following the induction of oxidative stress in IMR90 fibroblasts, revealed that oxidative damage does not appear to affect the rate of telomere erosion, or the frequency of large-scale telomeric deletion. The data are more consistent with the view that premature senescence does not arise as a consequence of accelerated telomere erosion, but instead more likely results from stochastic DNA damage across the rest of the genome. The analysis of telomere dynamics following the induction of chromosome fragility, showed that telomere length in Seckel cell (SCK) fibroblasts were significantly different from those of untreated cells following treatments with aphidicolin with an increase in stochastic telomeric deletion. Whilst in MRC5 fibroblasts, the induction of the telomere fragility impacted on the upper to lower allele ratio, with a loss of the longer telomere length distributions. The stabilisation of G-quadruplex structures using the G-quadruplex ligand (RHPS4), together with ATRX knockdown, showed that an absence of ATRX sensitised cells to the ligand, but that the stabilisation of G-quadruplexes, did not significantly affect the telomere dynamics as determined using STELA. Taken together, the data presented in this thesis are not consistent with a role for oxidative stress, or the formation of G-quadruplex structures, in generating large-scale telomeric deletion; however telomeric mutational events may occur following the induction of chromosome fragile sites, specifically in the context of an ATR deficiency.

The first part of my thesis dealt with the physical mapping of human chromosome 2 employing the yeast artificial chromosome (YAC) cloning system. To generate a chromosome 2 YAC sublibrary, over 1,000 interspersed repetitive sequence (IRS)-PCR probes were generated and used to screen the CEPH midi YAC library and approximately 2,000 chromosome 2-specific midi YACs were identified. These YACs were divided into 223 YAC groups, i.e., sets of unordered overlapping YACs, and using publicly available contig analysis software, a tentative order of YACs within each YAC group could be established. To order YAC groups, the chromosome 2 YAC sublibrary was screened with 87 genetically mapped microsatellites and cytogenetically mapped expressed sequence tags (ESTs), and 44 YAC groups were localized along the genetic map of chromosome 2q. In addition, 16 known genes were physically linked with microsatellites within YAC groups, thus providing integration points for genetic, cytogenetic and YAC-based physical maps of chromosome 2q. In a subsequent step of the analysis, the chromosome 2 YAC mapping data created by the Whitehead Institute (WI)/MIT Genome Center were integrated into our dataset. The integrated dataset consisted of 240 YAC groups, of which 14 large groups containing both our and WL/MIT Genome Center YAC groups were located on chromosome 2q. These 14 groups consisted of 1,195 YACs, which will form the backbone for the construction of a complete YAC contig for human chromosome 2q.
The second part of my thesis dealt with the identification of genetic markers within or in the vicinity of NRAMP1, a candidate tuberculosis susceptibility locus. The human NRAMP1 gene was mapped to chromosome 2q35 by PCR analysis in a monochromosomal hybrid panel and by YAC contig analysis. Nine sequence variants and polymorphisms were identified within the NRAMP1 gene by single strand conformation analysis (SSCA), DNA sequencing and Southern analyses. Furthermore, two highly informative microsatellites, D2S104 and D2S173 were shown to be linked to NRAMP1 within a 1.5 Mbp YAC contig. Together, these markers provide molecular tools for further genetic analysis of inherited susceptibility to tuberculosis and related diseases of the macrophage.

The work described here involves the use of fluorescence measurements to investigate the intramolecular quadruplex formed by the human telomeric repeat. By monitoring the efficiency of energy transfer between fluorophores either side of the structure, it is possible to follow the opening of the quadruplex in real time. In many of the experiments described here this opening was induced by the addition of a strand complementary to the quadruplex-forming sequence, to form a conventional duplex structure. Initial studies focused on the rate of opening of the human telomeric intramolecular quadruplex using peptide nucleic acid (PNA), an artificial nucleic acid mimic with an unchanged back-bone. It was observed that, for micromolar PNA, the rate of opening was independent of the concentration of PNA. This is probably due to the rate of opening being limited by a rearrangement of the quadruplex-forming strand that must occur before hybridisation can take place. The activation energy of opening was obtained by repeating the opening at several temperatures. One reason for the choice of a fluorescence-based technique is the great sensitivity with which such measurements can be performed. This allows the use of much lower sample concentration than would be possible with other measurements, such as circular dichroism or UV absorbance. With the correct equipment it is possible to detect the emission from single fluorescent species. These sorts of measurements remove the averaging that occurs when measuring many molecules at the same time. The use of single molecule detection was applied to the study of the human telomeric intramolecular quadruplex, for which there are two published structures. Two species were found in solution, and molecular modelling was used to propose assignments of these to known structures for this quadruplex. Finally, the interaction between the human telomeric intramolecular quadruplex and a peptide-hemicyanine conjugate ligand was investigated. In the presence of a complementary DNA strand the quadruplex opens and forms a duplex. It was observed that in the presence of sodium ions the ligand slowed down this hybridisation in a manner that allowed the determination of the binding constant between the quadruplex and the ligand. This was repeated at several temperatures in order to obtain thermodynamic and kinetic parameters for binding and hybridisation respectively. Furthermore, in the presence of potassium the ligand was observed to have no effect on the rate of hybridisation. The results of these experiments were used to suggest a structural model for binding.

Telomeres, the nucleoprotein structures at the ends of linear chromosomes, maintain genomic stability by protecting chromosome ends from fusion, degradation, and processing by the DNA double-strand break repair machinery. Telomere shortening, which occurs naturally in somatic cells during aging, leads to cellular senescence or apoptosis. In contrast, germline cells and cancer cells acquire unlimited replicative potential by activating a telomere lengthening mechanism, generally via reactivating the enzyme telomerase reverse transcriptase. To date, drug development targeting cellular immortalization in cancer has focused on telomerase inhibition. However, in a subset of tumours and in vitro-immortalized cell lines, telomeres are maintained by homologous recombination-mediated pathways, termed alternative lengthening of telomeres (ALT). ALT tumours are expected to be refractory to anti-telomerase therapies, so the ability to rapidly and reliably screen for ALT status in tumour-derived cells is essential for guiding therapeutic strategies that target cellular immortalization. One characteristic of ALT-mediated telomere maintenance is the presence of extrachromosomal telomeric repeat-containing DNA circles (t-circles), which provide an attractive target for detection in screening applications. Current methods oft-circle detection require considerable amounts of cells, making them unsuitable for analysis of limited clinical samples. We optimized a screen for ALT status based on a novel technique of rolling circle amplification (RCA) oft-circles from extrachromosomal DNA extracts of human ALT cells. We demonstrate that RCA requires a much lower number of cells than previously established t-circle detection methods, and screening many samples can be performed in parallel, making RCA suitable for analyzing clinical samples. T-circles were reproducibly detected in human immortalized ALT cell lines, but not in telomerase-utilizing cell lines. In addition, ectopic over-expression of telomerase in an ALT cell line does not appear to affect t-circle formation. This suggests that presence of active telomerase within a cell does not inhibit all telomeric recombination reactions. The potential for RCA as a tool to screen tumour samples for ALT activity and the link between telomerase and ALT-based telomere lengthening mechanisms are discussed.

Activation of a telomere maintenance mechanism is a vital step in the development of most cancers and provides a target for the selective killing of cancer cells. Cancers can use either telomerase or Alternative Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition of either telomere maintenance mechanism can cause cancer cells to undergo senescence or apoptosis. Although telomerase inhibitors are undergoing clinical trials, on commencing this study very little was known about the role of ALT in cancer, what proteins were involved in its mechanism and regulation and how it could be targeted clinically. The primary aim of this thesis was to develop an assay for ALT suitable for examining archived tumour specimens and to begin using it to examine the prevalence and clinical significance of ALT in cancer. This assay and gene expression analysis was also used to identify genes that are involved in or associated with the activation of the ALT mechanism, to contribute towards the overall goal of an ALT cancer therapy. The ALT mechanism involves recombination mediated replication and ALT cells have a marked increase in a range of recombinational events specifically at their telomeres. Presumably, as a consequence of this the telomere lengths of ALT cells are very heterogeneous and on average long. This can be detected by terminal restriction fragment (TRF) Southern analysis, which has been used previously as the definitive test for ALT activity. However, TRF analysis requires intact genomic DNA and is unsuitable for tumour specimens which are commonly archived by paraffin embedding. Another hallmark of ALT is ALT-associated PML bodies (APBs) which are the subset of PML bodies that contain telomeric DNA. Work done in this study to consolidate APBs as a hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31 telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three APB[+]/telomerase[+] cell lines identified here had an order of magnitude lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be functionally linked to the ALT mechanism and contain the recombination proteins that are thought to be involved in the ALT mechanism. This study, in collaboration with Dr W-Q Jiang, strengthened this functional link by demonstrating that loss of ALT activity (as determined by TRF analysis) coincided with the disruption of APBs. The detection of APBs was developed into a robust assay for ALT in archived tumour specimens using a technique of combined immunofluorescence and telomere fluorescence in situ hybridisation. It was demonstrated that the APB assay concurred exactly with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF analysis gave unequivocal results. The APB assay may be a more appropriate technique in the case of tumour specimen heterogeneity, which may explain why the APB assay was able to give definitive results when TRF analysis was equivocal. We demonstrated that intratumoral heterogeneity for ALT does exist and this could explain why about 3% of tumours in this study were APB[+] but with more than a ten-fold reduction in the frequency of APB[+] nuclei. This study also made the novel discovery of single stranded C-rich telomeric DNA inside APBs which potentially could be used to make the APB assay more suitable for routine pathology laboratory use. The APB assay was used to show that ALT is a significant concern for oncology. ALT was utilised in approximately one quarter of glioblastoma multiforme (GBM), one third of soft tissue sarcomas (STS) including three quarters of malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with these ALT[+] tumours had poor survival; median survivals were 2 years for ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and 5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also just as aggressive as their ALT[-] counterparts in terms of grade and patient outcome. ALT status was not found to be associated with response to chemotherapy in osteosarcomas or survival in STS. ALT was however, less prevalent in metastatic STS. The APB assay was a prognostic indicator for GBM and was correlated with three fold increased median survival in GBM (although this survival was still poor). ALT was more common in lower grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had an identical median age at diagnosis to that reported for secondary GBM. It is discussed that these data indicate that ALT was indirectly associated with secondary GBM and is possibly an early event in its progression from lower grade astrocytoma. This is relevant because secondary GBM have distinct genetic alterations that may facilitate activation of the ALT mechanism. Putative repressors of ALT could explain why this study found that ALT varied among the different STS subtypes. ALT was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which have a high prevalence of telomerase activity and a reduced need for a telomere maintenance mechanism (low cell turnover), respectively. A yeast model of ALT predicts that one of the five human RecQ helicases may be required for ALT. Using the APB assay to test for the presence of ALT in tumours from patients with known mutations in either WRN or RECQL4 it was demonstrated that neither of these RecQ helicases is essential for ALT. Although p53 and mismatch repair (MMR) proteins have been suggested to be possible repressors of ALT, there was no apparent increase in the frequency of ALT in tumours from patients with a germline mutation in p53 codon 273 or in colorectal carcinomas that had microsatellite instability and thus MMR deficiency. Also contrary to being a repressor of ALT but consistent with its ability to interact with a protein involved in the ALT mechanism, the MMR protein MLH1, was demonstrated to be present in the APBs of an ALT[+] cell line. To further test for genes that may be involved in the ALT mechanism or associated with its activation, RNA microarray was used to compare the gene expression of 12 ALT[+] with 12 matched telomerase[+] cell lines; 240 genes were identified that were significantly differentially expressed (p<0.005) between the ALT[+] and telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly differentially expressed after adjusting for the estimated false positive rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be relevant to the ALT[+] tumours and Western analysis indicated that DRG2 and MGMT levels were down-regulated after activation of ALT and up-regulated after activation of telomerase, whereas SATB1 protein levels appeared to be up-regulated after immortalisation but to a higher degree with activation of ALT compared to telomerase. Since lack of MGMT is known to be a determinant of temozolomide sensitivity in GBM, the possibility that ALT and the APB assay could be used to predict temozolomide sensitivity is discussed. The microarray data was consistent with MGMT expression being suppressed by EGF (p < 0.05), indicating that caution may be needed with combining EGFR inhibitors with temozolomide in ALT cancers. One ALT[+] cell line which did not express MGMT had TTAA sequence in its telomeres. This could possibly have resulted from mutations due to lack of MGMT expression and a possible role for MGMT in the ALT mechanism is discussed. Further analysis of the microarray data identified two groups of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT, and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a bias towards genes with higher expression levels in ALT. This work has developed a robust assay for ALT in tumour specimens which was then used to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It has also identified genes that could possibly be molecular targets for the treatment of ALT[+] cancers.

Doctor of Philosophy
Activation of a telomere maintenance mechanism is a vital step in the development of most cancers and provides a target for the selective killing of cancer cells. Cancers can use either telomerase or Alternative Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition of either telomere maintenance mechanism can cause cancer cells to undergo senescence or apoptosis. Although telomerase inhibitors are undergoing clinical trials, on commencing this study very little was known about the role of ALT in cancer, what proteins were involved in its mechanism and regulation and how it could be targeted clinically. The primary aim of this thesis was to develop an assay for ALT suitable for examining archived tumour specimens and to begin using it to examine the prevalence and clinical significance of ALT in cancer. This assay and gene expression analysis was also used to identify genes that are involved in or associated with the activation of the ALT mechanism, to contribute towards the overall goal of an ALT cancer therapy. The ALT mechanism involves recombination mediated replication and ALT cells have a marked increase in a range of recombinational events specifically at their telomeres. Presumably, as a consequence of this the telomere lengths of ALT cells are very heterogeneous and on average long. This can be detected by terminal restriction fragment (TRF) Southern analysis, which has been used previously as the definitive test for ALT activity. However, TRF analysis requires intact genomic DNA and is unsuitable for tumour specimens which are commonly archived by paraffin embedding. Another hallmark of ALT is ALT-associated PML bodies (APBs) which are the subset of PML bodies that contain telomeric DNA. Work done in this study to consolidate APBs as a hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31 telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three APB[+]/telomerase[+] cell lines identified here had an order of magnitude lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be functionally linked to the ALT mechanism and contain the recombination proteins that are thought to be involved in the ALT mechanism. This study, in collaboration with Dr W-Q Jiang, strengthened this functional link by demonstrating that loss of ALT activity (as determined by TRF analysis) coincided with the disruption of APBs. The detection of APBs was developed into a robust assay for ALT in archived tumour specimens using a technique of combined immunofluorescence and telomere fluorescence in situ hybridisation. It was demonstrated that the APB assay concurred exactly with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF analysis gave unequivocal results. The APB assay may be a more appropriate technique in the case of tumour specimen heterogeneity, which may explain why the APB assay was able to give definitive results when TRF analysis was equivocal. We demonstrated that intratumoral heterogeneity for ALT does exist and this could explain why about 3% of tumours in this study were APB[+] but with more than a ten-fold reduction in the frequency of APB[+] nuclei. This study also made the novel discovery of single stranded C-rich telomeric DNA inside APBs which potentially could be used to make the APB assay more suitable for routine pathology laboratory use. The APB assay was used to show that ALT is a significant concern for oncology. ALT was utilised in approximately one quarter of glioblastoma multiforme (GBM), one third of soft tissue sarcomas (STS) including three quarters of malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with these ALT[+] tumours had poor survival; median survivals were 2 years for ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and 5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also just as aggressive as their ALT[-] counterparts in terms of grade and patient outcome. ALT status was not found to be associated with response to chemotherapy in osteosarcomas or survival in STS. ALT was however, less prevalent in metastatic STS. The APB assay was a prognostic indicator for GBM and was correlated with three fold increased median survival in GBM (although this survival was still poor). ALT was more common in lower grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had an identical median age at diagnosis to that reported for secondary GBM. It is discussed that these data indicate that ALT was indirectly associated with secondary GBM and is possibly an early event in its progression from lower grade astrocytoma. This is relevant because secondary GBM have distinct genetic alterations that may facilitate activation of the ALT mechanism. Putative repressors of ALT could explain why this study found that ALT varied among the different STS subtypes. ALT was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which have a high prevalence of telomerase activity and a reduced need for a telomere maintenance mechanism (low cell turnover), respectively. A yeast model of ALT predicts that one of the five human RecQ helicases may be required for ALT. Using the APB assay to test for the presence of ALT in tumours from patients with known mutations in either WRN or RECQL4 it was demonstrated that neither of these RecQ helicases is essential for ALT. Although p53 and mismatch repair (MMR) proteins have been suggested to be possible repressors of ALT, there was no apparent increase in the frequency of ALT in tumours from patients with a germline mutation in p53 codon 273 or in colorectal carcinomas that had microsatellite instability and thus MMR deficiency. Also contrary to being a repressor of ALT but consistent with its ability to interact with a protein involved in the ALT mechanism, the MMR protein MLH1, was demonstrated to be present in the APBs of an ALT[+] cell line. To further test for genes that may be involved in the ALT mechanism or associated with its activation, RNA microarray was used to compare the gene expression of 12 ALT[+] with 12 matched telomerase[+] cell lines; 240 genes were identified that were significantly differentially expressed (p<0.005) between the ALT[+] and telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly differentially expressed after adjusting for the estimated false positive rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be relevant to the ALT[+] tumours and Western analysis indicated that DRG2 and MGMT levels were down-regulated after activation of ALT and up-regulated after activation of telomerase, whereas SATB1 protein levels appeared to be up-regulated after immortalisation but to a higher degree with activation of ALT compared to telomerase. Since lack of MGMT is known to be a determinant of temozolomide sensitivity in GBM, the possibility that ALT and the APB assay could be used to predict temozolomide sensitivity is discussed. The microarray data was consistent with MGMT expression being suppressed by EGF (p < 0.05), indicating that caution may be needed with combining EGFR inhibitors with temozolomide in ALT cancers. One ALT[+] cell line which did not express MGMT had TTAA sequence in its telomeres. This could possibly have resulted from mutations due to lack of MGMT expression and a possible role for MGMT in the ALT mechanism is discussed. Further analysis of the microarray data identified two groups of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT, and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a bias towards genes with higher expression levels in ALT. This work has developed a robust assay for ALT in tumour specimens which was then used to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It has also identified genes that could possibly be molecular targets for the treatment of ALT[+] cancers.

Telomeres are DNA-protein complexes that help protecting the end of linear chromosomes. They consist of repetitive DNA, in mammals the repeat unit is the hexanucleotide TTAGGG, these repeats span 5-20 kb. Under normal conditions in somatic cells, telomeres get shorter with every population doubling until they reach a critical length and then, the cell enters a checkpoint called senescence or M1 where it stops dividing. If the cell escapes senescence and continues dividing with further telomere shortening, it reaches a second checkpoint called crisis or M2. Crisis is characterized by telomere dysfunction leading to genomic instability that can end with cell death. However, some cells achieve to maintain telomere length by activating a telomere maintenance mechanism (TMM). The presence of a TMM is a hallmark of cancer cells. Two TMM have been described in human cells, one is the through the enzyme telomerase, which is active in 85% of cancers, and the second is a homologous recombination (HR) based mechanism called Alternative Lengthening of Telomeres (ALT) active in 15% of cancers. The evidence that the ALT pathway relies in HR was the observation that sequences can be copied from one telomere to another in ALT+ but not in telomerase+ cells and that several genes involved in HR are necessary for ALT progression. The ALT pathway is not the only event involving HR at telomeres. It has been shown that the human herpesvirus 6 (HHV-6) can integrate into human telomeres. Interestingly, HHV-6 possesses perfect telomeric repeats within its genome. The proposed mechanism for integration if through HR between the telomeric repeats present in the virus with the human telomere repeats. The aim of this work is to unravel the molecular mechanism underlying the ALT pathway and HHV-6 integration. The data obtained will contribute to the understanding of HR in human telomeres.

In Barrett's oesophagus, the metaplastic tissue displayed clonal growth characterised by very short homogeneous telomere profiles compared to the adjacent normal tissue. Short telomeres and fusions were also detected in normal dermis and melanocytic naevi with evidence of clonal telomere fusion events. Such events can result in clonal expansion, which could confer selective advantage for other abnormalities that may derive neoplastic progression. These data are consistent with the view that telomere dysfunction in vivo may drive large-scale genomic instability of the type observed in early-stage neoplasia.

The terminal regions of 7q and 12q show a high level of similarity (96 %) for at least 2 kb of DNA adjacent to the telomere. In addition, the start of the telomere repeat arrays coincide with respect to the flanking DNA at these two chromosome ends. This suggests that a duplication has occurred and that the proximal region of the telomere was involved. The 7q and 12q telomere-adjacent regions contain a diverged subterminal repeat that suggests multiple rearrangements have been involved in the formation of the modern 7q and 12q telomeres. A polymorphic telomere (the 'Nitru' telomere) has been isolated, and is located at 16p and 16q. The telomere-adjacent sequence from the Nitu telomere is present on 18 autosomes, but it is only adjacent to a telomere repeat array at these two chromosome ends. The Nitu telomere is present in approximately 8 % of individuals from various populations. Analysis of the interspersion patterns of TTAGGG and variant repeats, at the proximal end of these three autosomal telomeres, indicates that there is no similarity between them. The 7q and 12q telomeres can be distinguished easily despite the similarity in the flanking DNA. The Nitu telomere repeat arrays are different again, although it is not possible to determine the location (16p or 16q) by the telomere interspersion pattern or the 200 bp of DNA immediately adjacent. This may suggest that the Nitu telomere duplication is relatively recent. Analysis of single alleles at the 12q and Nitu telomeres demonstrates a high level of variation, suggesting a high level of turnover. The allelic variation within these autosomal telomeres indicates that the major mechanism involved in the turnover of repeats is intra-allelic.

Rtel1 est une hélicase qui a été identifiée comme un facteur essentiel pour maintenir les télomères longs et le génome stable chez la souris. Chez l'homme, des mutations germinales dans RTEL1 ont été trouvées chez les patients atteints du syndrome de Hoyeraal-Hreidarsson (HHS), une forme grave de la dyskératose congénitale. Cependant, le mécanisme selon lequel cette protéine agit dans les cellules humaines reste en grande partie inconnu. Pour étudier la fonction de RTEL1 sur le métabolisme des télomères nous avons réduit l'expression de RTEL1 par ARN interférent dans plusieurs lignées de cellules humaines et analysé la longueur des télomères par quantitative-FISH. Nos résultats montrent que la dérégulation de RTEL1 induit un raccourcissement des télomères uniquement dans les cellules avec de très longs télomères et surexprimant la télomérase. Nous démontrons également que l'absence de RTEL1 provoque une altération du complexe de shelterin au télomères: l'augmentation des niveaux de TRF2 et la diminution de POT1. La surexpression de la portion OB fold de POT1 peut restaurer le raccourcissement des télomères causé par le knockdown de RTEL1. Ceci indique que RTEL1 peut jouer un rôle important dans la stabilité du 3' sortant et l'accessibilité de la télomérase. Nous constatons également un impact de RTEL1 sur le métabolisme de l'ARN non codant télomérique TERRA. En effet, la diminution de RTEL1 réduit la quantité totale de TERRA présente dans le noyau et en particulier de TERRA associé aux télomères. Nous constatons que ce nombre réduit de TERRA est causé par sa dégradation, donc nous proposons que RTEL1 a un rôle dans la stabilisation de TERRA aux télomères
Rtel1, regulator of telomere elongation helicase 1, was discovered as an essential factor for telomere length maintenance and genomic stability in mice. In humans, germline mutations in RTEL1 have been found in patients with Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenita. However, the precise mechanism of action of the protein in human cells remains largely unknown. To investigate the function of RTEL1 in human telomere metabolism we used a knockdown approach by specific siRNAs and quantitative-FISH to measure telomere length after depletion of RTEL1 in different cancer cell lines. Our results show that down-regulation of RTEL1 induces shortening of telomeres only in cells with very long telomeres and high telomerase activity. We also demonstrate that upon depletion of RTEL1 there is a different stochiometry of shelterin proteins at telomeres: increased levels of TRF2 and decreased levels of POT1. Importantly, the overexpression of the POT1 OB fold can rescue the shortening of telomeres caused by the knockdown of RTEL1 indicating that RTEL1 may play an important role in the stability of the overhang and in its accessibility to telomerase. We also find an affect of RTEL1 on Telomeric non-coding RNA (TERRA) metabolism. Indeed, depletion of RTEL1 in human cell lines reduces the total amount of TERRA present in the nucleus and in particular of telomere-associated TERRA. Moreover, we find that this reduced number of UUAGGG repeats is caused by TERRA degradation, therefore we propose that RTEL1 has a role in stabilizing TERRA at telomeres

Telomeres cap the end of eukaryotic chromosomes and prevent the natural end of a chromosome from being recognised as a double-stranded DNA break. Dysfunctional telomeres may trigger replicative senescence, or fuse with other telomeres or with non-telomeric DNA breaks. The length of a telomere plays a key role in telomere function. Relatively little is known about how telomeric chromatin influences telomere length and function. A number of studies in mammalian cells have identified a handful of chromatin remodelling proteins and the chromatin marks they deposit in telomere length regulation. However such examples at human telomeres are scarce. The primary aim of this thesis was to investigate whether the chromatin structure of a telomere is a determinant of its length in human cells. Two approaches were taken to address this issue: Firstly, the chromatin structure of telomeres of differing lengths were directly analysed by measuring enrichment of histone modifications known to be prominent at telomeres in other model organisms. Secondly, selected chromatin remodelling proteins were studied to determine whether they play a role in telomeric chromatin structure and telomere length. Single Telomere Length Analysis (STELA) provides a high resolution method to measure telomere length distributions at individual chromosome ends. STELA assays were previously designed for the 2p, 9p, 11q, 12q, 16p, 17p and 18q telomeres. An allele‐specific STELA assay has also been designed for the XpYp chromosome end. In this study novel telomere and telomeric allele‐specific qPCR assays were developed for the same chromosome ends. These qPCR assays, when used in conjunction with ChIP provide a tool for analysing telomeric chromatin structure at individual chromosome ends. Applying this ChIP‐qPCR approach alongside STELA allows any correlations between telomeric chromatin structure and telomere length to be identified. This approach suggested differences in telomeric chromatin structure between telomeres of different lengths in telomerase‐positive HT1080 fibrosarcoma cells. Shorter HT1080 telomeres were less abundant in H3 and TRF1 and also had lower levels of H4K20me3 and, to a lesser extent, H3K4me3 compared to longer telomeres. Differences in chromatin structure were not observed between telomeres of different lengths in telomerase negative MRC5 fibroblasts. Changes in chromatin structure were observed at individual telomeres/telomere alleles were observed between actively proliferating cells and in cells undergoing senescence. Telomeric enrichment of H3 and TRF1 as well as the histone methylation marks H3K4me3, H3K9me3 and H4K20me3 were reduced in senescent cells. The degree of chromatin structural change as the cells entered senescence differed between chromosome ends. This highlights the benefits of using the telomere-specific ChIP-qPCR approach over the more traditional ChIP-dot blot assays which would not be able to differentiate between the chromatin structure of different chromosome ends. To identify roles for chromatin remodelling proteins in telomere length maintenance siRNA mediated knockdown of selected chromatin remodelers was performed in a clonal population of HT1080 cells followed by STELA analysis. RNAi-depletion of the histone methyltransferase (HMTase) III EHMT2 resulted in an increase in very short 17p telomeres whereas loss of another HMTase, DOT1L caused a divergence in the 17p telomere length distribution suggesting the presence of two subpopulations of cells each with differing telomere length distributions. Subtle changes in mean telomere length was observed after siRNA mediated knockdown of the HMTases MLL and EZH2, the histone deacetylases (HDACs) HDAC1 and SIRT6, the ATP dependent chromatin remodelling complex subunit BAF155 and the H3.3 histone chaperone DAXX. However due to certain limitations of the RNAi screen the validity of these observations is questionable and more work would have to be performed to confirm whether these chromatin remodelers have an effect on telomere length. Finally, dramatic telomere shortening was observed in a keratinocyte holoclone population after siRNA mediated knockdown of DAXX at number of chromosome ends. Prolonged depletion of DAXX also caused an increase in telomere‐to‐telomere fusions. A similarly dramatic loss in telomere length was seen in these cells after knockdown of EHMT2.

Replicative senescence describes the irreversible growth arrest that primary human fibroblasts undergo when cultivated in vitro and represents one example of a biological process known as cellular senescence. Cellular senescence is associated with organismal ageing and functions an important tumour suppressive mechanism. A conditionally immortalised human mammary fibroblast cell line, HMF3A, has been developed with the aim of determining the precise molecular basis of telomere-independent cellular senescence. HMF3A cells constitutively express hTERT, the catalytic component of human telomerase, and a temperature sensitive non-DNA-binding mutant of Simian Virus 40 large T (LT) antigen. At the permissive temperature, 33.5C, HMF3A cells grow normally but at the non- permissive temperature, 39.5C, LT antigen is inactivated and the cells undergo a rapid and synchronous irreversible growth arrest. The stringency of HMF3 A growth complementation has enabled me to functionally analyse the pathways implicated in the induction of senescence in these cells. It was concluded that inactivation of the p53 pathway was sufficient to overcome the conditional HMF3A growth arrest. However, expression of a pl6iNK4a insensitive cvciin D1-CDK4R24C fusion construct indicated that the pRb pathway was not critical for the induction of this process. I subsequently identified three novel markers of telomere-independent cellular senescence by cDNA microarray analysis (namely, AKR1B1, CDH13 and UBE2C), and three potential regulators of senescence by an RNA interference screen (namely, NEUROD2, TARBP1 and RRM2). Finally, I detected a novel functional activity of the Adenovirus El A 13S splice variant that is not shared with the El A 12S splice variant. This activity enabled 13S El A, but not 12S El A, to bypass the conditional HMF3A growth arrest. The genes identified using these independent experimental approaches constitute novel markers of senescence and, upon validation of these findings, may provide prognostic and/or diagnostic value in the context of tumorigenesis.

Cellular senescence is an irreversible program of cell cycle arrest triggered in normal somatic cells in response to a variety of intrinsic and extrinsic stimuli including telomere attrition, DNA damage, physiological stress and oncogene activation. Finding that inactivation of the pRB and p53 pathways by SV40-LT antigen cooperates with hTERT to immortalize cells has allowed us to use a thermolabile mutant of SV40-LT to develop human fibroblasts where the cells are immortal if grown at 34oC but undergo an irreversible growth arrest within 5 days at 38oC. When these cells cease dividing, senescence-associated-β-galactosidase (SA-β-Gal) activity is induced and the growth-arrested cells have many features of senescent cells. Since these cells growth-arrest in a synchronous manner, I have used Affymetrix expression profiling to identify the genes differentially expressed upon senescence. This identified 816 up- and 961 down-regulated genes whose expression was reversed when growth arrest was abrogated. I have shown that senescence was associated with activation of the NF-κB pathway and up-regulation of a number of senescence-associated-secretory-proteins including IL6. Perturbation of NF-κB signalling either by direct silencing of NF-κB subunits or by upstream modulation overcame growth-arrest indicating that activation of NF-κB signalling has a causal role in promoting senescence. I also applied a retroviral shRNA screen covering ~10,000 genes to the same cell model. Overlapping with the microarray data revealed particularly interesting targets, such as LTBP3 and Layilin. Finally, I profiled micro-rna expression. 15 of the top micro-rnas down-regulated upon senescence were chosen to express in the HMF3A system. 6 of them were able to bypass the growth-arrest. In conclusion, my work has uncovered novel markers involved in senescence as well as identifying that both activation of p53 and pRb pathway result in activation of NF-κB signalling which promotes senescence. Both results lead to a better understanding of senescence and its pathways.

Telomere instability was investigated at the proximal ends of human telomeres in normal and abnormal cells, with the aim to identify the frequency and types of mutations underlying telomere repeat turnover. Analysis of the interspersion patterns of telomere and variant repeat types at the proximal ends of the 12q and Xp/Yp telomeres in human pedigrees gave a germline mutation frequency of 0.6% per telomere per gamete over the proximal 1kb of the telomere repeat array. No somatic telomere mutations were identified in normal fibroblast cells, but the upper limit for the mutation frequency was estimated as 7.468xlO-3 per cell. Each of 7 germline mutation events involved increases or decreases in small numbers of repeats. These events can be explained by intra-allelic mutational mechanisms, such as replication slippage and unequal sister-chromatid exchange. Localised telomere instability was associated with the CTAGGG variant repeat type in 6/7 germline mutations. Approximately 15% of sporadic colon cancers and the majority of tumours from patients with hereditary non polyposis colon cancer (HNPCC) are caused by defects in genes involved in the mismatch repair pathway. Such defects result in an accummulation of mutations, particularly at microsatellite loci. A high mutation frequency was observed in the 12q and Xp/Yp telomere repeat arrays in colon cancers and was particularly associated with tumours showing microsatellite instability. The observed tumour mutations also involved increases or decreases in blocks of like-repeat types and can be explained by intra-allelic mutations. In one tumour, a complex telomere mutation was provisionally identified and may represent an inter-allelic mutation event. No telomeric mutations were observed in the first kilobase of the 12q and Xp/Yp telomeres in cell-lines derived from patients suffering from the premature ageing disorder Werner syndrome. The lack of telomere mutations in Werner syndrome cells is discussed, with respect to overall telomere stability.

The DNA immediately flanking the human Xp:Yp telomere exhibits a high level of sequence polymorphism and strong linkage disequilibrium, resulting in a limited number of highly diverged haplotypes. The sequence divergence suggests that these haplotypes are ancient. Orthologous sequences in chimpanzees and gorillas are more diverged than between synonymous sequences at other loci. Balancing selection may have contributed to the maintenance of the highly diverged haplotypes flanking the human Xp:Yp telomere and may explain the high sequence divergence in closely related species. A system has been developed to assay the distribution of telomere and variant repeats within the proximal 120 repeats of Xp:Yp telomeres to create a telomere map. The distributions of these repeats is highly polymorphic with estimated heterozygosities in excess of 99%. The mutation rate underlying this variation was measured directly as 6.25x10.;-3 per gamete. Alleles were grouped by similarities in their telomeremaps, these groups of alleles also share the same haplotype in the telomere flanking DNA. This suggests that these alleles have evolved along haploid lineages and that the predominant mutational mechanisms influencing the evolution of sequences at the proximal end of this telomere must be of an intra-allelic nature. Comparison of alleles suggests that most of the differences could be accounted for by small localised replication-slippage like events. Analysis of the chimpanzee and gorilla sequences orthologous to the human Xp:Yp telomere revealed that a telomere was not present at the same location in these species. Instead, two small interstitial blocks of telomere-like repeats were present in gorilla and an array of chimpanzee and gorilla specific subterminal satellite was present in chimpanzees. Therefore, the location of the Xp:Yp telomere is unique to the human lineage and may therefore be relatively new in evolutionary terms.

Telomere length has been shown to be a critical determinant of T cell replicative capacity and in vivo persistence in humans. We evaluated telomere lengths in virus-specific T cells to understand how they may both shape and be changed by the maintenance of memory T cells during a subsequent virus re-infection or reactivation. We used longitudinal peripheral blood samples from healthy donors and samples from a long-term HCV clinical interferon therapy trial to test our hypotheses. To assess T cell telomere lengths, I developed novel modifications to the flow cytometry fluorescence in situ hybridization (flowFISH) assay. These flowFISH modifications were necessary to enable quantification of telomere length in activated, proliferating T cells. Adoption of a fixation-permeabilization protocol with RNA nuclease treatment prior to telomere probe hybridization were required to produce telomere length estimates that were consistent with a conventional telomere restriction fragment length Southern blot assay. We hypothesized that exposure to a non-recurring, acute virus infection would produce memory T cells with longer telomeres than those specific for recurring or reactivating virus infections. We used two acute viruses, vaccinia virus (VACV) and influenza A virus (IAV) and two latent-reactivating herpesviruses, cytomegalovirus (CMV) and varicella zoster virus (VZV) for these studies. Combining a proliferation assay with flowFISH, I found telomeres in VACV-specific CD4 + T cells were longer than those specific for the recurring exposure IAV; data which support my hypothesis. Counter to my hypothesis, CMV-specific CD4 + T cells had longer telomeres than IAV-specific CD4 + T cells. We assessed virus-specific CD4 + T cell telomere length in five donors over a period of 8-10 years which allowed us to develop a linear model of average virus-specific telomere length changes. These studies also found evidence of long telomere, virus-specific CD45RA + T cell populations whose depletion may precede an increased susceptibility to latent virus reactivation. I tested the hypothesis that type I interferon therapy would accelerate T cell telomere loss using PBMC samples from a cohort of chronic hepatitis C virus patients who either did or did not receive an extended course of treatment with interferon-alpha. Accelerated telomere losses occurred in naïve T cells in the interferon therapy group and were concentrated in the first half of 48 months of interferon therapy. Steady accumulation of CD57 + memory T cells in the control group, but not the therapy group, suggested that interferon also accelerated memory turnover. Based on our data, I present proposed models of memory T cell maintenance and impacts of T cell telomere length loss as we age.

G-quadruplex structures are found in important regions of the eukaryotic genome, such as telomeres and regulatory sequences of genes, and are likely to play important roles in regulation of biological events. The significant structural differences with duplex DNA make quadruplex DNA a very attractive target for anticancer drug design. The purpose of this study is to explore conformational space in a series of heterocyclic cations to discover novel structural motifs that can selectively bind and stabilize specific G-quadruplex arrangements. A variety of biophysical techniques such as thermal melting experiments, biosensor surface plasmon resonance, circular dichroism, fluorescence displacement assay and mass spectrometry were employed to evaluate the affinity of the compounds and their recognition properties. The screening of the molecules allowed the identification of not only selective G-quadruplex ligands but also potential quadruplex groove binders. These results can be useful for the development of new efficient telomerase inhibitors which are endowed with pharmacological activity.

Telomeres are physical ends of chromosomes consisting of (TTAGGG)n DNA sequence and a specialized set of proteins that protect chromosomal ends from degradation and from eliciting DNA damage response. These specialized set of proteins, known as shelterin, directly bind to telomeric DNA. In addition, some DNA double-strand break (DSB) repair proteins such as, DNA-PKcs and KU70/80, play active roles in telomere maintenance. Mouse knock-out experiments have revealed that deletion of either DNA-PKcs or Ku70/80 resulted in elevated levels of telomeric fusion, indicative of dysfunctional telomeres. Artemis protein is involved in DNA DSB repair through non-homologous end joining (NHEJ) and it is phosphorylated by DNAPKcs. Human cells defective in Artemis have been identified and shown to be radiosensitive and patients with an Artemis defective gene suffer from radiosensitive severe-combined immune deficiency syndrome (RS-SCID). Mouse cells defective in Artemis have elevated levels of telomeric fusion. We have demonstrated in this thesis that Artemis defective human cell lines show a mild telomeric dysfunction phenotype detectable at the cytological level. The nature of telomere dysfunction phenotype appears to be similar to that observed in DNAPKcs defective cells as exemplified by the presence of IR induced chromatid telomeric fusions. We have also shown that (a) DNA damage occurring within the telomeric DNA is difficult to repair or irreparable in older cells and that (b) Artemis defective older cells show higher proportion of DNA damage at telomeres than their normal counterparts. Finally, we have demonstrated that inhibition of DNA-PKcs causes (a) an increase in telomeric fusions in Artemis defective cell lines relative to both normal cell lines after inhibition and Artemis cell lines before inhibition and (b)elevated levels of DNA damage at telomeres following exposure of cells to radiation relative to both irradiated normal cells exposed to a DNA-PKcs inhibitor and irradiated Artemis defective cells but not exposed to the DNA-PKcs inhibitor. These results suggest that the effects of Artemis and DNA-PKcs on telomeres are cumulative. We have also performed (a) experiments to examine telomere function in Artemis defective cell lines after knocking down DNA-PKcs levels by RNAi and b) preliminary experiments to knock-down Artemis in DNA-PKcs defective cells. Taken together, our results suggest that the Artemis defect causes mild telomere dysfunction phenotype in human cells.

Cellular immortalisation is currently regarded as an essential step in malignant transformation and is consequently considered a hallmark of cancer. Acquisition of replicative immortality is achieved by activation of a telomere lengthening mechanism (TLM), either telomerase or the alternative lengthening of telomeres (ALT), to counter normal telomere attrition. However, a proportion of malignancies are reported to be TLM-negative. The lack of serial untreated malignant human tumour samples over time has made it impossible to examine telomere length over time and hence determine whether they are truly TLM-deficient, or whether this is the result of false-negative assays. Here we describe a subset (11%) of high-risk neuroblastomas (NB) that lack evidence of any significant TLM activity despite a 51% 5-year mortality rate. Two NB cell lines derived from such tumours proliferated for 500 population doublings (PDs) with ever-shorter telomeres (EST). The EST cells had exceptionally long and heterogeneous telomere lengths as measured by terminal restriction fragment analysis and telomere fluorescence in situ hybridisation. Both cell lines were telomerase negative during culturing and did not have elevated markers of ALT or associated gene mutations. The telomeres of these cells shortened by 80 and 55 bases/PD, consistent with telomere attrition due to normal cell division, but did not reach senescence after 500 PDs in culture. This is conclusive evidence that cells from highly malignant, lethal tumours are able to undergo continuous proliferation in spite of an EST phenotype. The EST phenotype was rescued by activation of telomerase (via transduction with hTERT expression constructs) or ALT (spontaneous occurrence of a nonsense TP53 mutation, followed by spontaneous activation of ALT after 100 PDs). We also found that NB EST cells are very sensitive to topoisomerase I inhibitors indicating the potential to target the EST phenotype with topoisomerase I inhibitors in high-risk NB.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

Replicative exhaustion of endothelial cells (ECs) contributes to the pathogenesis of age-related vascular disorders, including atherosclerosis and impaired wound healing. Conversely, abnormal proliferation of ECs underlies the development of EC-derived malignancies, such as haemangioblastoma and angiosarcoma. The central objective of this thesis was to delineate mechanisms that regulate the replicative lifespan of human ECs and molecular alterations that occur during immortalisation of ECs. The gradual shortening of telomeres (chromosome-end structures) is one mechanism that restricts the replicative lifespan of human ECs. Telomere shortening initiates an irreversible growth arrest or senescence through activation of a TP53-mediated DNA damage response. Expression of the cyclin-dependent kinase inhibitor, p16INK4a, is also increased and reinforces senescence via the retinoblastoma pathway. Overexpression of telomerase reverse transcriptase (hTERT) reconstitutes telomerase activity and extends the lifespan of human ECs, but is not sufficient for immortalisation. The current study demonstrated that p16INK4a repression by promoter methylation was a frequent event during immortalisation of hTERT-transduced bone marrow ECs (BMECs), occurring in 5 of 12 clones. Repression of p16INK4a concurred with the development of recurring chromosomal aberrations, which appeared to be a consequence of telomere dysfunction and chromosome fusions. Loss of p16INK4a and the development of a complex karyotype were associated with a more transformed phenotype in hTERT-immortalised BMECs. The investigations described in this thesis were the first to associate loss of p16INK4a expression with the accumulation of chromosome aberrations. Repression of p16INK4a in only a subset of immortal BMECs provided impetus for investigating whether there was a functionally analogous defect in the hTERT-immortalised BMECs that retained p16INK4a expression. In normal human cells, oncogenic Ras upregulates p16INK4a and induces senescence independently of telomere shortening. This thesis demonstrates that the immortal BMECs that retained p16INK4a expression had a defective response to oncogenic Ras, which may have contributed to the immortalisation of these cells. Whole genome and proteome analyses identified additional alterations in gene copy number and protein expression specific to p16INK4a-positive or -negative immortal BMECs. Overall, these investigations provide new insight to the potential consequences of p16INK4a repression during carcinogenesis and describe novel molecular alterations that occur during immortalisation of human ECs.

Telomeres are protective structures at the end of eukaryotic chromosomes essential for indefinite cell proliferation. Their disruption causes activation of DNA repair pathways, growth arrest and/or cell death. In normal cells telomere shortening during cell division has been proposed to act as a tumor suppressor mechanism to block the proliferation of cells at risk of undergoing malignant transformation. Overcoming this proliferative block by activating a mechanism to maintain telomeres is a necessary requirement for unlimited proliferation and tumor progression. Human cells have two mechanisms for telomere maintenance: a more common one based on telomerase and a rarer one based on recombination called ALT.
Here we report the isolation of an immortal human cell line that maintains short telomeres in the absence of biologically active telomerase and key features of active ALT. Our results suggest that the mechanisms of telomere maintenance in human cells may be more diverse than previously thought and have important implications for the development of anti-cancer strategies based on the inhibition of telomere maintenance.
Due to widespread distribution of telomerase in human tumors and its absence in most normal cells, telomerase is the main target of these anti-cancer strategies. However, targeting telomerase per se or in combination with anti-cancer drugs is not sufficient to trigger rapid cell death of tumor cells. On the other hand, disturbances in telomere capping do not require telomere shortening to induce growth arrest and may act more quickly. Our goal was to investigate the feasibility of a new approach based on the combination of telomere destabilization and chemotherapeutic drugs. Our results show that interfering with telomere maintenance enhances the susceptibility of human tumor cells to anti-cancer drugs independently of their telomere lengths and mechanisms to maintain them.
Finally, given the involvement of telomeres in maintaining genomic stability, we investigated the mechanism by which mutations in the telomerase RNA subunit contribute to autosomal dominant dyskeratosis congenita, a premature ageing disease associated with mutations in the telomerase holoenzyme. Our data strongly indicate that the clinical manifestations of this disease may be caused by telomere shortening due to haploinsufficiency of telomerase activity and provide a direct correlation between disturbances in telomere length maintenance and human disease.

Human telomeres can be maintained by the enzyme telomerase, which catalyses the addition of telomere repeats, or by the Alternative Lengthening of Telomeres (ALT) mechanism, which is recombination based. Recently, knockout mouse cell lines have indicated that epigenetic modifications associated with telomeric and subtelomeric chromatin, including DNA methylation, Histone 3 Lysine 9 (H31K9) trimethylation and Histone 4 Lysine 20 (H4K20) trimethylation, play an important role in influencing the choice of telomere maintenance mechanism. In this thesis, the levels of these modifications were studied in telomeric chromatin and in the telomere adjacent chromatin of the short arm of the human sex chromosomes (XpYp) in a panel of six human cell lines utilising different telomere maintenance mechanisms. In marked contrast to mouse models, no relationship was found between the level of any of these modifications and telomere maintenance pathway. This may indicate that the role such marks play at telomeres differs between humans and mice. Additionally, the level of chromatin modifications associated with binding of 53BP1 was not affected by the telomere maintenance mechanism used in human cell lines. Similarly, H3K9 and H4K20 trimethylation adjacent to the MS32 minisatellite did not show ALT-specific alterations despite the high level of instability at this locus in ALT positive cells. Subtelomeric DNA methylation in the mouse has been shown to have a dynamic relationship with telomere length. In the current study, this relationship was investigated in telomerase positive human cells using a DNA methylation sensitive variant of the Single Telomere Length Analysis (STELA) technique. No relationship between telomere length and DNA methylation at XpYp was found in telomerase positive cells. Additionally, ectopic expression of telomerase in primary human fibroblasts did not alter subtelomeric DNA methylation. However, evidence suggests that elongated telomeres can prevent loss of subtelomeric DNA methylation associated with replication in normal human cells.

Telomerase activity allows normal cells to achieve immortality, which is one of the hallmarks of cancer. It is required to maintain telomere length to protect chromosome ends from being recognised as damaged DNA. The protein TCAB1 is necessary for telomerase trafficking to telomeres, while telomeric protein TPP1 is essential for its interaction with telomeres. However, the mechanisms regulating these proteins are poorly understood. This thesis identifies TPP1 as a novel disease-causing gene in humans. A mutation in the ‘TEL patch’ (the surface area of TPP1 that interacts with telomerase) is likely to have caused short telomeres in a family with inherited bone marrow failure. We demonstrated that other amino acids within the TEL patch, F209 and S210, are also vital for the recruitment of telomerase to telomeres. Mutational analysis demonstrated that phosphorylation of S255 of TPP1, possibly by ATM, regulates the function of TPP1 in recruiting telomerase to telomeres. Phosphorylation of S64 of TCAB1 was hypothesised to regulate its function in telomerase localisation to telomeres; while the involvement of phosphorylation of this residue remains undetermined, we demonstrated that this region of the protein is important. Furthermore, S491 of TCAB1 emerged as a residue that is potentially involved in regulating telomerase recruitment to telomeres. Additionally, this thesis also provided the first evidence for an association between nuclear actin polymerisation and telomerase recruitment to telomeres. Inhibition of actin polymerisation or nuclear import disrupted telomerase recruitment, suggesting a novel role for nuclear structural integrity in this process. Collectively, these data increase our understanding of the regulation of telomerase recruitment to telomeres by the proteins TCAB1, TPP1, and actin. This understanding will inform new strategies for development of cancer therapeutic drugs and for developing better diagnostics and treatments for telomere biology disorders.

Telomeres are repetitive sequences located at the ends of human chromosomes. During DNA replication, DNA polymerase is unable to fully replicate telomeric DNA causing a progressive reduction in telomere length with each cell division. Consequently, telomeres shorten with age and when telomere length reaches a critical length the cell becomes dysfunctional or senescent. Increasing numbers of senescent cells results in reduced organ function and telomere length has been associated with age-associated diseases including cardiovascular disease and cancer. Telomere length is highly heritable and genome-wide association studies have identified several loci that associate with telomere length. Interestingly, two of these loci do not contain genes with known roles in telomere maintenance. In this study, CRISPR/Cas9 genome editing was used to knockout telomere length associated genes in human induced pluripotent stem cells (hiPSCs) with the ultimate aim of definitively linking a novel telomere length associated gene to telomere maintenance. CRISPR/Cas9 was used to create hiPSCs with no telomerase activity by knocking out TERT, which encodes the catalytic subunit of telomerase. Telomere length was maintained in control iPSCs, but reduced by approximately 1% per day during extended culture of TERT knockout hiPSCs. Flow cytometric analysis of mutant hiPSCs revealed that they continued to express markers of pluripotency but had increased expression of a differentiation marker. Next, CRISPR/Cas9 was used to generate mutations in the candidate telomere maintenance genes ACYP2 and TSPYL6, however, no difference in telomere length was observed after extended culture of hiPSCs carrying mutations in either gene. In conclusion, CRISPR/Cas9 genome editing was successfully used to generate mutant hiPSCs for TERT, which resulted in telomere shortening and for two candidate telomere maintenance genes, which had no effect on telomere length. Further analysis will be required to determine which gene mediates the association with telomere length at the ACYP2 locus.

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.

Polychlorniated Biphenyls (PCBs), a group of 209 individual congeners, are ubiquitous environmental pollutants and classified as probable human carcinogens. Hallmarks of aging and carcinogenesis are changes in telomerase activity and telomere length. I hypothesize that PCBs modulate telomerase activity and telomeres via interference in gene regulation and generation of reactive oxygen species (ROS) resulting in the dysregulation of cell growth. To explore this possibility, I exposed human skin keratinocytes (HaCaT) to a synthetic airborne PCB mixture (CAM) and individual congeners, i.e. PCB28, PCB52, PCB126 and PCB153. To mimic the chronic human exposure to PCBs and the slow process of carcinogenesis, a long term exposure period of 48 days and beyond was employed. All PCB congeners and CAM reduced telomerase activity, telomere length and cell growth. Among all PCBs, PCB126 had the most pronounced effect with reduction in telomerase activity, telomere length, hTERT and hTR gene expression and cell growth, while increasing TRF1 & TRF2 gene expression. PCB126 elicited an increase in CYP1A1 mRNA, CYP1A1 activity, DHE and DCFH oxidation levels from days 6 to 48, suggesting that increased ROS might be a causative factor for the reduction in telomerase activity and telomere length. However, transduction with hTERT and hTR subunits partly rescued telomerase activity, while treatment with PEG-catalase did not rescue telomerase activity suggesting that telomerase subunits play an important role on PCB126 induced effects on telomerase activity and telomere length. Since cells with shortened telomeres may escape crisis through telomerase reactivation, PCB126 treatment was continued until day 90. A change in growth behavior was observed from day 54 to 90, with cells recovering the proliferation rate, and increasing c-Myc, hTERT, and hTR gene expression level, re-activating telomerase activity and re-elongating telomere length. TRF1 & 2 gene expression started to decrease after day 66. From day 78, no increase in CYP1A1mRNA and its activity as well as CYP1B1, ALDH3A1, UGT1A1 and AhRRmRNA was observed suggesting that the AhR response pathway may have been altered. This study shows for the first time that PCBs initially reduce telomerase activity, telomere length, and cell growth, and can later lead to telomerase re-activation, telomere lengthening and increased cell growth with modulation of the AhR receptor pathway. This observation has broad implications for chronic PCB exposure scenarios.

Telomere shortening is a major mechanism to induce telomere uncapping and thus to signal growth arrest and/ or apoptosis and can be caused by different mechanisms, one of which is damage to DNA, to which telomeres appear to be particularly sensitive. Contradictory data exists on the relationship between conventionally used chemotherapeutic drugs and the telomere/ telomerase complex. The aim of the work described in this thesis was to determine whether or not damage to telomeres played a significant role in the cytotoxic action of the anti-cancer drugs cisplatin and etoposide. Two cell lines were used with either short (neuroblastorna cell line SHSY5Y) or long (lymphoblastic T cell line 1301) telomeres. Cytotoxic effects of the drugs were assessed by growth inhibition assays and measurement of apoptosis and cell cycle progression by flow cytometry. Etoposide caused readily detectable DNA strand breakage and led to formation of nuclear foci of phosphorylated histone y-H2A. X. Cisplatin treatment did not induce strand breaks after initial drug exposure but strand breaks and DNA damage foci were detected after further incubation. For cells with either long or short telomeres, no detectable changes in total telomere length or overhang length were observed before apoptosis became manifest. Preferential occurrences of single strand breaks in the G-rich strand of telorneres were not found. Through the development of a dual staining method it was established that drug-induced histone H2A. X foci did not colocalise to the telomeres. Telomerase was transiently activated by lower concentrations of etoposide and its activity decreased only after onset of apoptosis. Taken together, the results show no indication that telorneres and/ or telomeric damage play any preferential role as signal transducers towards apoptosis and/ or growth arrest in either of these cell lines. Also, the protective function of telornerase &-I - seems to be telomere independent. The data are consistent with a model of druginduced growth arrest and apoptosis being triggered by damage elsewhere in the genome.

While the latent genome of most Herpesviruses persists as a nuclear circular episome, previous research has suggested that Human Herpesvirus 6 (HHV-6) may integrate into host cell chromosomes, and be vertically transmitted in the germ-line. Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DRR) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DRL), we established a hypothesis that during latency, the HHV-6A and HHV-6B genome integrates into the telomeres of human chromosomes through homologous recombination with the n(TTAGGG) viral repeats, and the integrated virus can be induced to lytic replication. We sought, first, to definitively illustrate the in vitro and in vivo integration of HHV-6A and HHV-6B. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6A and Molt3 cell line by HHV-6B, the virus integrated into telomere of chromosomes. Next, peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per ml of blood. FISH confirmed that HHV-6 DNA co-localized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, while the integration site was identical among members of the same family. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome through the germ-line [inherited HHV-6 (iHHV-6)]. Amplification and sequencing of the HHV-6A and more recently HHV-6B viral-chromosome junction identified DRR integrated into the telomere directly adjacent to the subtelomere of the chromosome. After mapping the DRR of iHHV-6, we subsequently focused on determining if the DRL was present in the integrated genome and whether the remaining telomere sequence of the chromosome was extended beyond the DRL. Southern hybridization of PCR amplified HHV-6 integrated cell lines and iHHV-6 patients PBMCs indicate the presence of DRL within the integrated viral genome. Therefore, the genomic structure of the iHHV-6 is as follows: chromosome-subtelomere-(TTAGGG)5-41-DRR-U-DRL-(TTAGGG)n. During latent integration, no circular episomes were detected even by PCR. However, trichostatin-A treatment of PBMCs and in vitro integrated HEK-293 cells induced the reactivation of iHHV-6 from its latent integrated state. We demonstrated the induction of integrated iHHV-6 with trichostatin-A lead to the excision of the integrated genome and generation of the U-DR-U junction which signifies circularization and/or concatemer formation of the viral genome through rolling-circle replication. Taken together, the data suggests that HHV-6A and HHV-6B are unique among human herpesviruses: they specifically and efficiently integrate into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

Short, repetitive G-rich DNA sequences present at telomeres are synthesized by telomerase, a ribonucleoprotein consisting of a catalytic subunit, the telomerase reverse transcriptase, TERT, and an integrally associated RNA, TR. Human TERT (hTERT) can repetitively reverse transcribe its short RNA template, acting processively to add multiple telomeric repeats onto the same DNA substrate. We investigated if threshold levels of telomerase activity and processivity are required to maintain telomere length and/or function and immortalize human cells with limited lifespan. Specifically, we assessed hTERT variants with mutations in several motifs implicated in processivity, namely the N terminus (E79A, E90K), RT motif 1 (I624M), the 'Insertion in Fingers' domain (V791Y), the C 'catalytic center' motif (L866Y), the E 'primer grip' motif (W930F) and the C terminus (∆1047-1056 and ∆1107-1118). The N-terminal and motif 1 hTERT mutants did not reveal any interesting phenotypes in vitro or in cells. On the other hand, the remaining variants, except L866Y, displayed a substantial decrease in processivity. Despite the presence of short telomeres in cells expressing these low processivity telomerase variants, only W930F could immortalize limited lifespan human cells. We demonstrate that limiting levels of DNA synthesis on the order of 20% of wild-type, and extension of as few as three telomeric repeats displayed by W930F are sufficient to maintain functional telomeres and immortalize limited lifespan human cells. The hTERT-C-terminal mutants likely could not immortalize cells due to synthesis of only 2 or less telomeric repeats. V791Y could not maintain telomere function due to a failure to localize to telomeres. On the other hand, L866Y displayed a 2-3 fold increase in proccessivity compared to wild-type telomerase. Cells expressing this mutant displayed telomere elongation followed by heterogenous telomere lengths and an increase in short telomeres, and fragile sites at telomeres accompanied by telomere trimming, indicating that processivity levels above that displayed by wild-type telomerase lead to telomere replication stress. These results suggest that telomere function and length, and immortalization in human cells are regulated by telomerase enzyme processivity.
Des courtes séquences répétées et G-riches d'ADN présentes aux télomères sont synthétisées par télomérase, une ribonucléoprotéine constituée d'une sous-unité catalytique, 'telomerase reverse transcriptase' ou 'TERT', et un ARN associé nommé 'TR'. TERT humain (hTERT) peut diriger de façon répétitive la transcription inverse de son ARN, agissant processivement en ajouteant de multiples répétitions télomériques sur le substrat d'ADN. Nous avons étudié si des niveaux limites d'activité ou de processivité de télomérase sont nécessaires pour maintenir la taille ou la fonction des télomères et pour immortaliser des cellules humaines possédant une durée de vie limitée. Plus précisément, nous avons évalué plusieurs variants de hTERT avec des mutations dans des motifs impliqués dans la processivité, incluant l'extrémité N-terminale (E79A, E90K), le motif 1 du Reverse Transcriptase (RT) (I624M), le domaine 'Insertion in Fingers' (V791Y), le motif C (L866Y), le motif E (W930F) et l'extrémité C-terminale (Δ1047-1056 et Δ1107-1118). Les mutations dans le terminus N et le motif 1 de hTERT n'ont pas révélées de phénotypes intéressants. Les autres variants, sauf L866Y, ont demontré une diminution substantielle des niveaux de processivité. Malgré la présence de télomères courts dans les cellules exprimant ces variantes de processivité faibles, seul W930F pouvait immortaliser les cellules. Nous démontrons que le niveau de synthèse d'ADN de l'ordre de 20% de hTERT sauvage, et l'extension de seulement trois répétitions télomériques par W930F sont suffisants pour maintenir des télomères fonctionnels et immortaliser les cellules. Les variants avec des mutations dans le terminus C ne pouvaient pas immortalizer les cellules dues à la synthèse de seulement 2 ou moins de répétitions télomériques. V791Y ne pouvait pas maintenir la fonction des télomères en raison d'une incapacité à se localiser aux télomères. D'autre part, L866Y a demontré une augmentation des niveaux de proccessivité de 2-3 fois par rapport à la télomérase sauvage. Les cellules exprimant ce mutant ont presenté un rallongement des télomères, suivi de télomères de tailles hétérogènes et une augmentation du nombre de télomères courts, accompagné d'une augmentation de sites fragiles aux télomères et de télomères tronqués, tout ce qui indique que des niveaux de processivité plus élevés que ceux du type sauvage mènent à des difficultés réplicatives aux télomères. Ces résultats suggèrent que la fonction et la taille des télomères, et l'immortalisation des cellules humaines sont réguléss par la processivité de l'enzyme télomérase.

Cosmid CY29 is derived from a DNA library prepared from the human-mouse hybrid cell line 3E7, which contains multiple Y chromosomes. Sub-clone 29C1, derived from CY29, has been found to define a hypervariable locus within 23kb of the human pseudoautosomal telomere. This has been assigned the locus name DXYS14. The clone has contributed to a genetic map of the human pseudoautosomal region, and has been used to study properties of the adjacent telomere. This thesis describes an analysis of sequences at this locus. Maps of the short arm subtelomeric regions of three human sex chromosomes are presented. In addition, by following inheritance of 290 homology through selected pedigrees it has been possible to obtain data on subtelomeric regions of seven other sex chromosomes. From this data it is shown that a region containing 29C1 homologous sequence and sequence proximal to it are duplicated on some telomeres. Thus any individual may have two, three or four copies of 29C1 homologous sequence in total, with one or two on each sex chromosome. In addition, restriction fragments containing 29C1 homology vary in size due to variation in copy number of a minisatellite type repeat. Sequence data from three such fragments shows blocks of imperfect 31bp GC rich repeat units in variable numbers in a head to tail array. The consensus sequence of these repeats show some similarity to other, reported minisatellite sequences. However when used as a probe, a DXYS14 consensus repeat oligomer detects other hypervariable loci only weakly, even at low stringency. It appears therefore that the DXYS14 repeat represents a separate family of minisatellites present in one or two blocks per haploid genome, and isolated within 23kb of the human XY telomere. In one pedigree studied, a novel 29C1 homologous restriction fragment is shown to have arisen during male meiosis, probably by change in size of the repeat block. Sequence and mapping data indicate that the 29C1 repeat block and sequence proximal to it shows characteristics normally associated with a low methylation island. This region does not appear to be transcribed however, and in the light of knowledge of DNA structure around locus DXYS14, a gene at this site seems unlikely. Sequences homologous to 29C1 are found in a range of vertebrates, with strong homology in some primates yet none in others. The implications of these observations are discussed.

Le matériel génétique contenant l'information des cellules humaines se présente sous forme de chromosomes linéaires dont l'extrémité est protégée par une structure appelée télomères. Les télomères correspondent à une séquence d'ADN répétée, recouverte de protéines spécifiques, qui permettent aux cellules d'étiqueter l'extrémité de leurs chromosomes afin de les différencier des cassures internes de l'ADN nécessitant une réparation. Ainsi, ils jouent un rôle prépondérant dans la protection du génome. Les chromosomes sont organisés et compartimentés dans le noyau de la cellule. Cette organisation est primordiale, la proximité des chromosomes à la membrane nucléaire qui délimite ce noyau est essentielle pour de nombreuses fonctions régulatrices du génome, comme l'activation et la répression des gènes contenant les informations. A chaque division cellulaire, cette organisation est perdue après le désassemblage de la membrane nucléaire et la condensation de la chromatine qui va permettre de correctement répartir les chromosomes entre les cellules filles. Après la division, les noyaux des cellules filles se reforment, la membrane nucléaire est rétablie, et les chromosomes sont repositionnés comme dans la cellule mère. Ce mécanisme de mémoire spatiale est encore inconnu mais est vital au maintien de la stabilité du génome. Une large proportion de télomères sont ancrés à la membrane nucléaire en fin de division, et y restent durant la reformation du noyau. Le laboratoire s'intéresse à cette association afin de caractériser son rôle pendant cette phase clé du cycle cellulaire. Nous cherchons à comprendre ce fonctionnement chez les cellules normales et les cellules de patients atteints de pathologies associées au vieillissement accéléré. Ce projet de thèse à pour but de comprendre l'impact d'une déformation de la membrane nucléaire sur le matériel génétique, et sur l'intégrité des télomères qui protègent l'information génétique. Nous utilisons des techniques de pointe de microscopie, et de biologie cellulaire et moléculaire afin de mieux comprendre le lien entre l'organisation du noyau et le maintien de la stabilité du génome
The material that contains genetic information of human cells consists in linear chromosomes. The extremities of chromosomes are protected by a specific structure called telomeres. Telomeres are made of repeated DNA sequence, covered by special proteins that prevent cells to recognize extremities of their chromosomes as internal DNA break, thus not to perform unnecessary repair that will result in genome instability. Therefore, telomeres play a major role in genome protection. Chromosomes are spatially organized in the cell nucleus. This organization is important as positioning of chromosomes in the nucleus ensures proper regulatory functions of the genome, such as activation or repression of genes. During the cell division process, this organization is lost after nuclear membrane disassembly and the condensation of DNA, to allow correct segregation of chromosomes between daughter cells. After cell division, the nuclei of daughter cells are reformed, and nuclear membrane is reconstructed. The chromosomes are then relocated as in the mother cell. This mechanism of spatial memory is not well understood yet, but is key to maintain stability of the genome. A large proportion of telomeres are anchored to the nuclear membrane at the end of mitosis, and stay during nuclear envelope reformation. Our laboratory focuses on characterizing the role of telomere anchoring during this important phase of cell cycle. In particular, we want to understand this mechanism in normal cells and cells from patients with premature aging disease. This thesis aims to understand the impact of nuclear envelope abnormalities on the genetic material, in particular on telomere integrity, as telomeres protect genetic information. Here, we use microscopy approaches and techniques of molecular and cellular biology to better understand the link between nuclear organisation and genome stability maintenance