Browsing by Subject "Telomeres"
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Item Duplication and Diversification of Arabidopsis thaliana Telomerase RNP Components(2012-02-14) Cifuentes-Rojas, CatherineTelomerase is a highly regulated ribonucleoprotein complex that stabilizes eukaryotic genomes by replenishing telomeric repeats on chromosome ends. Defects in telomerase RNP components involving the catalytic subunit TERT or the RNA template TER lead to stem cell-related diseases such as dyskeratosis congenita and idiopathic pulmonary fibrosis, while inappropriate telomerase expression is a rate-limiting step in carcinogenesis. In this study we report the discovery of a novel negative regulatory mechanism for telomerase that stems from duplication and diversification of key components of the telomerase RNP in the flowering plant Arabidopsis thaliana. We show that Arabidopsis encodes three distinct TERs: TER1, TER2 and a processed form of TER2 termed TER2S. Although all three RNAs can serve as templates for telomerase in vitro, in vivo they have different expression patterns, assemble into distinct RNPs with different protein binding partners, and play opposing roles in telomere maintenance. The TER1 RNP is analogous to the telomerase enzyme previously described in other eukaryotes, but the TER2 RNP is a negative regulator of telomerase activity and telomere maintenance in vivo. Furthermore, we demonstrate that the Protection Of Telomeres (POT1) paralogs in Arabidopsis (POT1a, POT1b and POT1c) are novel TER binding proteins. This finding is striking because in yeast and vertebrates, POT1 is an essential component of the telomere capping complex and functions to distinguish the chromosome terminus from a double-strand break. Thus, our data argue that Arabidopsis POT1 proteins have migrated off of the chromosome terminus and onto the telomerase RNP, indicating that duplication and diversification of Arabidopsis telomerase may be the end result of the co-evolution of the TER and POT1 RNP components. Additionally, given the dire consequences of misregulating telomerase in human cells, our discovery of a novel negative regulatory mechanism for telomerase in plants strongly suggests that additional modes of telomerase control remain to be elucidated in vertebrates.Item Genetic and Molecular Studies of Endometrial Cancer(2010-11-02T18:10:45Z) Akbay, Esra; Castrillon, Diego HEndometrial cancer, which develops from the inner lining of the uterus, is the most common cancer of the female reproductive tract. Endometrial cancer is comprised of two epidemiologic, molecularly and genetically different subtypes known as type I and type II. Because of its clinical significance and aggressive behavior, my research focused on type II endometrial cancer. Type II endometrial cancer is associated with advanced age and TP53 mutations, which suggests a link between telomeres and the development of type II tumors. Telomeres and other markers of telomere status in type I and type II tumors were analyzed, short telomeres are observed in both tumor types. However, only type II tumors are associated with critical telomere shortening in the adjacent, morphologically normal epithelium. This suggests that telomere shortening contributes to the initiation of type II, but not of type I tumors. To further explore this hypothesis, 5th generation telomerase knockout mice with critically short telomeres were analyzed and a distinctive endometrial lesion that resembles the in situ precursor of type II carcinomas, endometrial intraepithelial carcinomas were observed. Subsequently, the role of dysfunctional telomeres in endometrial cancer development was investigated in the mouse by conditionally inactivating Pot1a, a key component of the shelterin complex that stabilizes telomeres in endometrial epithelium. Inactivation of Pot1a by itself does not stimulate endometrial carcinogenesis. However, simultaneous inactivation of Pot1a and p53 results in endometrial intraepithelial carcinoma-like lesions by 9 months and metastatic tumors in 100% of the animals by 15 months. These tumors are poorly differentiated endometrial carcinomas with prominent nuclear atypia, DNA damage, and aneuploidy, resembling human type II tumors. These studies thus lend support to the hypothesis that dysfunctional telomeres play a critical role in type II endometrial carcinogenesis.Item Human shRNA Library Screening to Dissect Pathways Involved In Telomerase Actions(2011-12-12) Hoshiyama, Hirotoshi; Shay, Jerry W.The minimal components of human telomerase are the human telomerase reverse transcriptase (hTERT) and the human telomerase template RNA (hTR). Although it is known that both components are minimally sufficient to reconstitute telomerase activity, the factors involved in any of the multiple steps of telomerase action such as telomerase assembly, telomerase recruitment to telomeres, and telomere extension/regulation are not well understood. There are a large numbers of proteins that have associations with telomerase, yet the functional roles of those in telomere maintenance and telomerase regulation are not well understood. Identifying novel proteins and pathways involved in any of these important telomerase-associated functions will be useful for identifying new targets for the development of novel inhibitors that block telomerase function in cancer cells. Therefore, my goal has been to develop methods to dissect these molecular pathways and identify functional factors involved in any step of telomerase actions. To accomplish this, I designed a selective screening system by exploiting lentiviral shRNA libraries and tetracycline inducible-hTERT cell lines that is hTR deficient but expressing mutant hTR. Thus, the overall strategy of the screening system may be considered a “synthetic rescue screen”. In brief this screen was set up to rescue cells from apoptotic death due to mutant sequence incorporation at telomeres by reducing the gene expressions with lentiviral shRNA libraries. This allows us to look a set of genes involved in pathways involved in functional aspects of telomerase actions, not based on structural association with telomerase. During the work, I have established multiple lines of inducible-hTERT cells to use in selective screening systems. I have also developed a method for rapid construction of high-complexity custom shRNA libraries for targeted screening and re-evaluate hundreds of primary candidate genes to identify smaller numbers of secondary candidate genes by removing false positives. In order to analyze the pooled shRNA screening result, I have developed a method for quantitative identification of half-hairpins from a pooled shRNA library based on the pGIPZ vector. Introducing multiplexing codes and refining sample preparation schemes resulted in the predicted ability to detect two-fold enrichments followed by massive parallel sequencing. Development of those methods allowed me to identify several candidate proteins, which may be involved in telomerase actions.Item Telomere Dynamics and End Processing in Mammalian Cells(2006-05-15) Sfeir, Agnel J.; Shay, Jerry W.Telomeres are repetitive DNA sequences that end in single-stranded 3' overhangs. With each cell division, normal human cells lose a small amount of telomeric DNA due to the end-replication problem and the action of an unidentified nuclease. In order for tumor cells to divide indefinitely, they maintain telomere length by expressing the enzyme telomerase. The end structure of mammalian telomeres is not very well understood. Two assays were developed using ligation and PCR amplification to identify the terminal nucleotides of both the C-rich and G-rich telomeric strands in human cells. The results showed that ~ 80 % of the C-strands terminate precisely in ATC-5', demonstrating that the nuclease resection of the C-strand post replication is specific for a single nucleotide. In contrast, the last base of the G-strand in normal human cells was less precise with 70% of the ends being TAG-3', TTA-3' or GTT-3'. An enrichment for the TAG-3' end was noted in cells that express telomerase. A series of nucleases were tested for their involvement in specifying the last base of C-strands and the results indicated that none of those nucleases were responsible for telomere-end resection. Inhibiting the normal function of most telomere binding proteins altered normal telomere function, however only one protein (POT-1) influenced last base specificity. Knocking down POT-1 in normal and tumor cells randomized the last base of the C-strand. These finding have important implications for the processing events that act on the telomere ends and they will help identify the nuclease that resects the chromosome ends. In the second part of this study, the dynamics of telomerase action in mammalian cells was examined. Using a PCR-based, single telomere-length measurement assay (STELA) we showed that telomerase adds an average of 250-nucleotides per end in one replication cycle. Cell cycle studies showed that while the telomeres on the Xp chromosome replicated in early S-phase their elongation by telomerase took place during late S/early G2 phase. Therefore, in mammalian cells telomerase action is not coupled to DNA replication. These studies will provide much needed information for exploiting our knowledge of telomere biology for telomerase-based therapeutic purposes.Item Telomere Dynamics in Arabidopsis thaliana(2014-11-06) Renfrew, Kyle BraytonTelomeres are the physical ends of eukaryotic chromosomes. Because chromosome ends resemble double?strand breaks, telomere binding proteins mask telomeres from DNA damage response machinery. Consequently, telomere protection physically blocks telomere replication by the unique ribonucleoprotein (RNP) reverse transcriptase, telomerase. Telomerase access to telomeres is strictly regulated in the cell, and thus telomeres vacillate in status from telomerase accessible and telomerase inaccessible states. Here, I report the mechanistic contributions of the telomerase accessory protein POT1a (Protection Of Telomeres 1) in Arabidopsis telomere dynamics. POT1a, one of three POT1 paralogs in Arabidopsis, is essential for telomere replication. My work revealed POT1a is an activator of telomerase and stimulates its enzymology. POT1a physically binds two telomere proteins, CTC1 and STN1, and all three proteins can associate with active telomerase in vivo. In contrast, POT1a competed with TEN1, a capping protein shown to negatively regulate telomerase activity, for an interface on STN1. Thus, POT1a contributes to telomere dynamics though its interactions with telomerase and telomere binding proteins. Additionally, I examined the function of TER2, a non-canonical telomerase RNA that negatively regulates the TER1 (canonical) telomerase RNP. Null mutations in ter2 result in mild telomere phenotypes. However, when ter2 mutation was combined with the loss of POT1a, pot1a ter2 double mutants exhibited severe rates of telomere shortening and early onset defects in plant morphology and development. Thus, POT1a and TER2 represent two distinct regulators of telomere maintenance in Arabidopsis thaliana. Lastly, I observed the consequences of POT1 gene duplication. Evolutionary analysis revealed POT1a post-duplication was under Darwinian selection pressure for non-synonymous changes in three amino acid sites. Reversion of these residues back to the ancestral (POT1b) state resulted in a reduced ability of these mutants to genetically complement the telomere maintenance defect of pot1a mutants. In addition, these mutants had a reduced affinity for CTC1 in vitro. Therefore, POT1a is under positive evolutionary selection for its role in telomere maintenance and its association with CTC1. In summary, my work has elucidated the contributions of POT1a to Arabidopsis telomere dynamics, and how these functions contribute to its role in promoting telomere maintenance.Item Telomere Position Effect in Human Cells(2003-04-01) Baur, Joseph Anthony; Shay, JerryTelomeres are tracts of repetitive DNA that cap the ends of linear chromosomes. Each time the chromosome is duplicated, a small amount of telomeric DNA is lost from the end due to factors inherent in the mechanism of DNA replication. The result is a net shortening of telomeres with each cell division, unless new repeats are synthesized through the action of the enzyme telomerase. Most human somatic cells lack telomerase activity and so continued cell division leads to telomere shortening. After a limited number of divisions (the "Hayflick limit"), it is believed that a few critically shortened telomeres trigger a state of growth arrest termed replicative senescence.Item Telomere Regulation in Arabidopsis thaliana by the CST Capping Complex and DNA Damage Response Proteins(2013-09-11) Boltz, Kara A.The ends of chormosomes are capped by telomeres, which distinguish the termini from damaged DNA. Paradoxically, DNA repair proteins are also required for telomere maintenance. How DNA repair pathways are regulated to maintain telomeres while remaining competent to repair DNA damage throughout the genome is unknown. In this dissertation, I used a genetic approach to investigate how critical components of telomerase and the telomere protein complex interact with the DNA damage response (DDR). In the flowering plant, Arabidopsis thaliana telomeres are bound by the CST (CTC1/STN1/TEN1) heterotrimer. Loss of any CST component results in telomere shortening, telomere fusions, increased G-overhang length and telomere recombination. To understand the phenotypes caused by CST deficiency, I examined telomeres from plants lacking CTC1 or STN1 and TERT or KU. My analysis showed that CST acts in a separate genetic pathway for telomere length regulation from both KU and TERT. Further, I found that KU and CST act in separate genetic pathways for regulation of G-overhang formation. These demonstrate that multiple pathways are used to maintain telomere length and architecture in plants. My study of the interaction of telomere components with the DDR revealed ATR promotes genome stability and telomere length maintenance in the absence of CTC1, probably by activating programmed cell death of stem cells with high amounts of DNA damage. I also found that poly(ADP-ribosylation) is not required for maintenance of Arabidopsis telomeres, in contrast to human telomeres. Finally, I found an unexpected connection between the DDR and telomerase. My research showed that ATR maintains telomerase activity levels. Further, induction of double- stranded DNA breaks in seedlings led to a rapid decrease in telomerase activity, which correlated with increased abundance of TER2, an alternate Arabidopsis telomerase RNA. I hypothesize that TER2 inhibits telomerase to prevent its inappropriate action at internal sites in chromosomes. These data reveal two ways that DDR pathways work in concert with telomerase to promote genome integrity.Item Telomeres and telomere binding proteins in Arabidopsis thaliana(Texas A&M University, 2004-09-30) Shakirov, Yevgeniy VitalievichTelomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary to prevent chromosome fusions and exonuclease attack. We found that telomere tracts in Arabidopsis are fairly uniformly distributed throughout a size range of 2-9kb. Unexpectedly, telomeres in WS plants displayed a bimodal size distribution with some individuals exhibiting 4-8 kb telomeres and others 2-5 kb telomeres. We also examined the dynamics of telomere tracts on individual chromosome ends. Following the fate of telomeres in plants through successive generations, we found that the shortest telomeres were typically elongated in the subsequent generation, while the longest telomeres were usually shortened. Thus, telomere length homoeostasis is achieved through intermittent telomerase action on shorter telomeres to attain an optimal size.Single-strand telomere binding proteins were also analyzed. Four major telomere binding protein complexes from cauliflower were identified and their DNA-binding properties characterized. The DNA-binding component of one of the complexes was purified and analyzed by mass-spectrometry. Peptide mass data was used to search for putative protein candidates from the Arabidopsis thaliana database. Additionally, two Arabidopsis genes, AtPot1 and AtPot2, were identified and characterized. The genes encode two single-strand telomeric DNA binding proteins. AtPot1 and AtPot2 proteins can homo- and heterodimerize in vitro. Pot1 protein predominantly localizes to the nucleolus, whereas Pot2 is exclusively nuclear. Plants over-expressing full-length Pot1 and Pot2 proteins had no obvious phenotype, while over-expression of P2DBD and P1?DBD caused moderate telomere shortening. Plants over-expressing P2DBD had severe morphological and reproductive defects, multiple chromosome abnormalities and aneuploidy. Over-expression of a chimeric protein DBD-P1?DBD led to rapid telomere shortening, confirming the involvement of Arabidopsis Pot proteins in telomere length maintenance. Intriguingly, telomerase in DBD-P1?DBD-EYFP plants is inactivated, suggesting that Pot proteins are also involved in regulation of telomerase activity. The analysis of Arabidopsis telomeres and telomere binding proteins will provide additional information towards understanding the role of the telomeric nucleoprotein complex in eukaryotic chromosome biology.