Browsing by Subject "Epistasis, Genetic"
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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.Item Narrowing of the SLES1 Internal Reveals Complex Epistatic Interactions in the Suppression of Autoimmunity(2010-05-14) Belobrajdic, Katherine Ann; Wakeland, Edward K.Sle1 is a potent susceptibility locus for spontaneous systemic autoimmunity derived from the NZM2410 mouse strain. The NZW-derived suppressive modifier locus, Sles1, specifically prevents the spontaneous loss in tolerance mediated by the B6.Sle1 congenic. Sles1 had previously been fine-mapped to a remarkably gene-rich region on murine chromosome 17 containing nearly 70 genes. A series of mouse strains were constructed with a variety of suppressive and non-suppressive variants of Sles1 on the B6.Sle1 genomic background which have revealed multiple layers of epistatic gene interactions within the Sles1 interval. Phenotyping of a truncated recombinant interval mapped the Sles1 phenotype to an approximately 638 KB segment, which combined with genomic and expression analysis, suggested Btnl2 and the H2 genes are strong candidates for Sles1. Finally, further characterization of the Sles1 interval has revealed an allele-specific and tissue-specific reduction of major histocompatibility complex (MHC) Class II molecules on the surface of B cells, as well as a possible role for follicular helper T cells in the development of Sle1-mediated autoimmunity. Understanding how Sles1 and other modifiers suppress systemic autoimmunity will reveal important insights for developing therapeutic strategies for systemic lupus eythematosus (SLE).Item The Structural Distribution of Epistasis in a Pair of Essential Metabolic Enzymes(December 2021) Nguyen, Thuy Ngoc-Thi; Hibbs, Ryan E.; Reynolds, Kimberly A.; Rice, Luke M.; Yu, HongtaoInteractions between proteins provide the basis for cells to perform metabolism, grow, divide, move, and appropriately respond to external stimuli. Because proteins do not act as independent entities, the genetic background influences the effect of a mutation in unexpected ways. This context-dependence of mutational effects is epistasis. Extensive progress has been made in our ability to identify epistasis between proteins. However, how the epistasis between a pair of proteins is distributed across the amino acid sequence is less clear. Previous work characterized this sequence-level epistasis between proteins that bind to form a physical complex. Until now, the structural pattern and magnitude of epistasis between pairs of mutations spanning interacting metabolic enzymes remained uncharacterized. In my dissertation work, I deeply examined the context dependence of mutations for two essential enzymes in the bacterial folate metabolic pathway, Dihydrofolate Reductase (DHFR) and Thymidylate Synthase (TYMS). To achieve this goal, I used deep mutational scanning assays on DHFR in the context of varying activities of TYMS. The result is a rigorous dataset with epistasis measurements over the entire amino acid sequence of DHFR. I found that the positions with the greatest magnitude of epistasis within the structure of DHFR lied at the active site. However, the sign of epistasis at the DHFR active site was dependent on whether TYMS was active. Beyond the active site, the distribution of positive epistasis among the positions of DHFR was also context- dependent on the state of TYMS. Therefore, we can think of the active site as a non-physical "interface" between protein pairs that do not form a physical complex but share an intermediate. The potential consequences of this dataset on the epistasis between DHFR and TYMS are profound. This dataset is fundamental towards our understanding of how epistasis mechanistically emerges in nonlinearities between catalytic activity in enzymes, protein abundance, and cellular growth rate. This experimental dataset is also necessary to credibly validate predictions of epistasis from models of statistical co-evolution.Item A Tool-Box for Quantifying the Relationship Between Gene Expression, Nutrient Conditions, and Cellular Growth Rate in Bacteria(2020-08-01T05:00:00.000Z) Mathis, Andrew David; Mishra, Prashant; Lin, Milo; Reynolds, Kimberly A.; Ross, Elliott M.A central aspect of the genotype to phenotype problem is relating changes in gene expression to cellular division (or growth rate). The relationship between gene expression and growth rate can be complex, nonlinear, and dependent on environmental conditions, however, most high-throughput studies condense this complexity into a single discrete measurement per gene. This greatly limits the utility of high-throughput screening data in applications like rational engineering of cellular systems, modeling of cellular behavior, and genetic screening for specific phenotypes. I developed a series of techniques, based on highthroughput CRISPR interference gene knockdowns, to more continuously quantify the effects of gene expression and nutrient condition on bacterial growth rate. These techniques allow high-throughput titration of gene expression, precise modulation of environment conditions, and corresponding quantification of growth rate, epistasis, and gene-by-environment interactions all in the same experiment. Using these techniques, I have demonstrated that epistasis can explain co-evolution between a pair of enzymes, that gene by environment interactions are often specific to certain gene expression regimes, and that the sign and magnitude of epistasis can be dependent gene expression levels. In sum, these techniques are an essential step towards developing predictive models that relate gene expression, nutrient condition, and growth rate.