Browsing by Subject "Telomerase"
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Item Cell cycle protein interaction with telomerase in a breast cancer culture system(Texas Tech University, 2001-08) Cook, T. KevinBreast cancer causes 40,000 deaths a year and is second only to lung cancer in deaths attributed to neoplastic disease. There is a known age and cell lineage dependence related to the development of the disease. It is vitally important to understand the manner in which this disease develops. One approach is to examine proteins involved in cell cycle regulation. Three such proteins are c-Myc, p53, and pRb. These proteins in conjunction with one another and telomerase perform critical functions within a cell. They have an intertwined pattern of activity. If these functions are not regulated, the cells can proliferate unchecked and give rise to cancer. Our study focused on the differences of protein levels and activity overtime between epithelial and stromal cells of the breast. The data appeared to show that stromal cells exhibit a tighter control over their cell cycle than their epithelial counterparts. Further, it would seem that c-Myc levels are inversely related to telomerase activity. This data points to more studies in the future.Item Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the HTERT Locus(2011-02-01T19:36:40Z) Wilson, Kimberly Anne; Porteus, MatthewThe ability to direct hTERT expression through genetic control or tunable regulatory factors would advance our understanding of the transcriptional regulation of hTERT, and also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and zinc finger nucleases (ZFNs) to target sequences at the hTERT promoter. We first explored expansions to the repertoire of sites that can be targeted by ZFNs and modifications of ZFN architecture to accommodate such sites. A ZFN is made of a zinc-finger DNA binding domain (ZFP) linked to the FokI nuclease domain by a short amino acid “inter-domain linker”. The general sequence motif of a ZFN target is 5’-(ZFN site1)-(6 bp spacer)-(ZFN site2)-3’ and each half-site is 5’-GNNGNNGNN-3’. Variations of this motif come in the forms of variable spacer lengths, extra basepairs in-between triplets, and the inclusion of non-GNN triplets. To explore these types of target sites, we created ZFN variants that contained different inter-domain linkers, lengthened inter-finger linkers, and DNA binding domains created through hybridizing the modular assembly and OPEN methodologies. We show that through altering ZFN architecture, target sites with 5-7-bp spacers and those with ANN, CNN, and TNN triplets can be efficiently recognized and cut by ZFNs. We then generated new ZFPs to five ZFN target sites with 5- or 6-bp spacers in the hTERT locus based on those findings and made ZFTFs by linking the ZFPs to the VP16 transcriptional activation domain. We were able to identify several active ZFTFs that demonstrate a dose-dependent response. The same ZFPs were also converted into ZFNs and screened in combinatorial pairs in cell-based single-strand annealing assays and gene targeting assays. These screening strategies have pinpointed several ZFN pairs that may be useful in genomic editing of the hTERT locus. Our findings provide guidelines for modifying ZFP architecture to a wider array of potential target sites for use in developing ZFTFs and ZFNs at the hTERT promoter, which may be applicable towards inheritable, telomerase-based diseases and answering basic science questions about hTERT transcriptional regulation.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 Telomerase Regulation in Arabidopsis thaliana(2012-10-19) Nelson, AndrewTelomeres form a nucleoprotein cap at the end of eukaryotic chromosomes. The telomere protein constituents repress the DNA damage response (DDR) and facilitate maintenance of terminal sequences by a specialized ribonucleoprotein complex called telomerase. In turn, factors involved in the DDR guarantee telomerase acts only in telomere homeostasis, and not at double-strand breaks (DSBs). Thus, the three pathways surrounding telomeres display incredible overlap and are immensely complex. Here, I report a novel regulatory pathway that limits telomerase action during DNA damage. Duplication of the telomerase RNA subunit (TER) in Arabidopsis has given rise to a TER that is not required for telomere homeostasis. Indeed, this TER, termed TER2, is a competitive inhibitor of TER1 RNP complexes. Exposure to genotoxic agents results in TER2 upregulation and a subsequent inhibition of telomerase activity. Using data from the 1,001 Arabidopsis genomes project, I determine that the TER duplication and inhibitory nature of TER2 is likely derived from a transposon-like element within TER2. This element is found throughout Brassicaceae, with at least 32 members in Arabidopsis lyrata. These findings highlight the complex and diverse mechanisms by which an organism will regulate telomerase action. Here I characterize two members of the A. thaliana POT1 gene family. Contrary to POT1a, these proteins appear to have derived unique ways to perform their roles in chromosome-end protection. POT1b may protect telomeres as part of a TER2 telomerase RNP complex, as telomere defects only appear in the absence of both POT1b and TER2. POT1c is also appears to provide for chromosome end protection and appears to compete with POT1a to regulate telomerase access to the G-overhang. Together, these proteins represent part of a critical telomere capping complex distinct from CST. Additionally, I describe a means for elucidating factors that regulate telomere addition at DSBs. This incredibly detrimental process, termed de novo telomere formation (DNTF), is toxic, and thus this work describes the first in depth characterization of DNTF in multicellular eukaryotes. In summary, my work describes several novel regulatory and protective mechanisms for keeping telomeres and DSBs distinct.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 Length Studies In Human Cancer Cells(2011-08-26T17:34:05Z) Buseman, Christen Marie; Shay, Jerry W.Telomeres consist of repetitive DNA sequences and their associated binding proteins, and serve to protect linear chromosome ends from being recognized as double stranded breaks in need of repair. The telomeres of most normal diploid cells shorten with every cell division until they reach a critically short length, at which time the cells undergo senescence or apoptosis. Cancer cells which have the ability to divide indefinitely must prevent their telomeres from becoming critically short, and the majority of cancer cells achieve this by upregulating telomerase. Maintaining telomere length involves regulating the dynamic between telomere shortening and telomere elongation. However, there are still many aspects of this dynamic regulatory process that are unknown. Many methods of telomere length assessment have been developed that utilize a variety of molecular techniques, but a major shortcoming of these methods is that they lack the ability to detect single short telomeres that are thought to trigger replicative senescence. Thus, the objective of this work was to develop an assay, named Universal STELA, which can generate an accurate distribution of telomere lengths on all chromosomes and allow for the study of the shortest telomeres in experimental settings. Universal STELA was first used to determine if cancer stem cells are susceptible to telomerase inhibition therapy because they have a larger fraction of shorter telomeres than non cancer stem cells. Cancer stem cells are thought to contribute to cancer metastasis and recurrence, and therapies like telomerase inhibition that target cancer stem cells may lead to more durable treatment outcomes. Universal STELA was next used to investigate regulation of telomerase action. C- and G-STELA were used to determine that telomerase activity is coupled to telomere replication, while C-strand fill-in is delayed until S/G2. Universal STELA was used to compare the rate of elongation of short, average and long telomeres when telomeres are shorter than their maintenance length.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 Telomere Specific Homologous Recombination in the Alternative Lengthening of Telomeres(2013-01-16) Whitington, Aric J.; Shay, Jerry W., Ph.D.; Wright, Woodring E., M.D., Ph.D.Over twenty years have passed since the discovery of telomerase-independent telomere maintenance, yet the precise details of the ALT mechanism remain a mystery. A growing body of evidence suggests that ALT cells maintain telomeres by homologous recombination (Reddel 2003 for Review). Groundbreaking work by Oliver Bechter demonstrated that ALT cells and telomerase-positive cells show no difference in the rate of general HR. This study fundamentally shaped our current concept of the ALT mechanism, implying that it involves preferential recombination of telomeric repeats. Since ALT seems to require proteins involved in normal HR, it follows that this telomeric recombination must be suppressed in telomerase positive or normal cells. However, to date there has been no direct evidence to support this hypothesis. Seeking to investigate the rates of telomere specific recombination, previous work in the Shay and Wright lab utilized the Tel-Tel vector. However, due to the method of integration only a limited number of clones could be analyzed and no statistically significant conclusions could be made. My work has focused on remedying this limitation. I have developed a strategy for integrating the Tel-Tel vector into a variety of host cell lines and generating a large number of distinct clones for each line. Using this strategy I was able to measure the average rates of telomeric HR for each cell line and provide the first direct evidence that ALT cells show distinctly elevated levels of increased telomere-specific HR. Additionally, I have constructed a control vector which functions in the same manner as Tel-Tel, differing only in that the telomeric repeats are replaced by non-telomeric repeat sequence. Using this vector (referred to as Mut-Mut) and the same incorporation method, I have demonstrated that there is no significant difference in the rates of general HR in ALT and telomerase positive cells. Finally, I used this novel ALT reporter as well as previously established methods to investigate some proteins that may play a central role in the ALT mechanism.