Title

Epigenetic analyses and modulation of nuclear reprogramming

Date of Completion

January 2009

Keywords

Biology, Genetics

Degree

Ph.D.

Abstract

This dissertation is composed of three lines of research regarding nuclear reprogramming. At first, expression profiles in deceased and live cloned pigs were compared to controls using microarray. Differentially expressed (DE) genes in deceased clones were potentially causing pathological anomalies such as dysregulated surfactant homeostasis in the lung. A large extent of DE genes in live clones demonstrates that live clones may be not merely the least affected animals but harbor magnified abnormalities, for example, delayed alveologenesis in the lung. Global DNA methylation analysis revealed hypomethylation in deceased clones whereas hypermethylation in live clones. Together, incomplete reprogramming by somatic cell nuclear transfer appears to contribute to dysregulated gene expression, resulting in pathogenesis in clones. ^ To determine the compatibility of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in cloned pigs, maternal lineages of clones were traced by mtDNA sequencing followed by expression profiling of nDNA- and mtDNA-encoded genes. While expression profiles of nDNA-encoded genes in clones were distinct from controls, mtDNA-encoded genes particularly in deceased clones showed an intermixed clustering. These results demonstrate that incomplete reprogramming adversely affects the gene expression of mtDNA. ^ To evaluate oxidative guanine as an alternative epigenetic modification, degree of guanine oxidation was measured in oxidative stress-induced mouse embryonic stem cells (ESC) Qualitative and quantitative analyses of oxidative guanine revealed that oxidative stress-induced ESCs harbored significantly increased level of 8oxoG Furthermore, the incidence of beating embryoid bodies differentiated from oxidative stress-induced ESCs was significantly increased, suggesting oxidative guanine has transmittable effects on cardiac-lineage specification after initiation of ESC differentiation. ^ In subsequent study, the reprogramming potential of carcinogens was evaluated. A putative reprogramming protocol was applied to mouse embryonic fibroblasts with 16 chemical candidates identified through function-oriented in silico high-throughput screening. From recovery day 2, alkaline phosphatase and surface specific embryonic antigen-1 positive colonies appeared at an efficiency of 0.02%. Expression profiling revealed that the colonies gained neuromuscular characteristics by partial reprogramming in addition to chemically induced adverse effects. ^ In conclusion, knowledge on epigenetic regulation of genes accumulated through these studies can possibly lead to precise regulation of nuclear reprogramming in the future. ^

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