Browsing by Subject "Telomere Shortening"
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Item Chemical Disruption of Wnt Signaling and Telomere Length Maintenance(2015-01-28) Kulak, Ozlem; White, Michael A.; Minna, John D.; Abrams, John M.; Lum, LawrenceA nearly universal feature of colorectal cancer (CRC) incidents is the presence of genetic alterations that promote deviant activation of the TCF/LEF transcriptional regulators. The TCF/LEF proteins control the transcriptional output of intercellular signaling mediated by the Wnt family of secreted ligands. Several chemical screening efforts devoted to disrupting deviant TCF/LEF activity have converged on two vulnerabilities in the Wnt pathway -- the poly-ADP-ribose polymerases, Tankyrase 1 and 2 (Tnks1 and 2) that control the threshold response levels to Wnt ligands, and the Wnt acyltransferase Porcupine that provides an essential fatty acyl adduct to all nineteen Wnt ligands. My thesis focuses on the chemical biology of one of these strategies -- the Tnks enzymes -- with the goal of understanding the strengths and limitations of drugging the Tnks proteins for achieving therapeutic goals in regenerative medicine and cancer. Given the previously assigned role of Tnks enzymes in telomere maintenance, I have also devoted considerable effort to understanding the cell biological effects of disrupting Tnks activity on telomere integrity. Finally, I mined a high-confidence collection of Wnt pathway inhibitors with previously unidentified mechanisms of action to identify novel small molecules that directly target the TCF/LEF transcriptional apparatus. This effort netted a chemical approach for disabling deviant transcriptional activity in CRC that is distinct from the one afforded by Tnks and Porcn inhibitors. Taken together, my thesis establishes a chemical toolkit for interrogating the inner workings of Wnt-mediated signaling and also reveals new avenues for disabling deviant Wnt responses in cancer and normal Wnt responses in tissue engineering.Item Defining Multiple Steps in Human Telomere End Processing(2012-07-10) Chow, Tan Hoi Tracy; Shay, Jerry W.Telomere overhangs are essential for chromosome end protection and telomerase extension, but how telomere overhangs are generated is unknown. Due to the classic end replication problem, leading DNA daughter strands are initially blunt while lagging daughters are shorter by at least the size of the final RNA primer, which historically is believed to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. Understanding the number and nature of the overhang processing events is crucial in establishing the roles of candidate proteins involved. We here define these steps in normal human cells. We show the final lagging RNA primer is positioned ~70-100 nt from chromosome ends (not at the extreme ends), and is not removed for ~1hr following replication. Therefore, the location of the RNA primer, rather than its size, is a primary driving force for telomere shortening. Moreover, we demonstrate that telomere end-processing occurs in two distinct phases following telomere duplex replication. During the early phase, which occupies 1-2 hours following telomere replication, several steps occur on both leading and lagging daughters. Leading telomere processing remains incomplete until late S/G2 when the C-terminal nucleotide is specified, referred to as the late phase. Furthermore, in human cancer cells under maintenance condition, telomerase extension is uncoupled from C-strand fill-in. These results uncover crucial mechanistic details of the DNA end-replication problem as well as several specific steps in telomere overhang processing. These results also indicate the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes. The findings and the methods developed will now provide the basis for examining candidate factors that may function to regulate particular steps in telomere length homeostasis with implications in both cellular aging and cancer.Item An Imaging Approach to Examine Telomere Dynamics and Regulation of Gene Expression with Aging(2020-08-01T05:00:00.000Z) Zhang, Ning; Xie, Yang; Danuser, Gaudenz; Shay, Jerry W.; Jaqaman, Khuloud; Siegwart, Daniel J.Telomeres are repetitive non-coding nucleotide sequences (TTAGGG)n capping the ends of chromosomes. Improved methods to measure the shortest (not just average) telomere lengths (TLs) are needed. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1-10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Therefore, demonstrating the regulatory roles of telomere length shortening on genes with accurate TL measurement will improve our understanding to the 3D genomic DNA landscape including telomeres. In this doctoral dissertation, I developed a user-friendly software for automatic electrophoresis gel quantification and contributed to developing the Telomere Shortest Length Assay (TeSLA), a technique that detects telomeres from all chromosome ends from <1 kb to 18 kb using small amounts of input DNA. Using cells with more TL information provided by TeSLA, I conducted an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. I examined two human genes, for which TPE-OLD has been described before, ISG15 (Interferon Stimulated Gene 15) and TERT (TElomerase Reverse Transcriptase). For both genes I found less interaction with the telomere on the same chromosome in old cells compared to young cells. Experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.