Date of Award

Fall 2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical Engineering

First Advisor

Chongli Yuan

Committee Chair

Chongli Yuan

Committee Member 1

Julie Liu

Committee Member 2

Doraiswami Ramkrishna

Committee Member 3

Ann Kirchmaier

Abstract

DNA methylation plays an essential role in various biological processes such as stem cell differentiation, imprinting, X-chromosome inactivation, etc. Increased DNA methylation levels have been associated with chromatin compaction leading to gene silencing. For example, abnormal DNA methylation is associated with silencing of tumor suppressor genes and is observed in the onset of tumorigenesis. There is evidence suggesting that not all methylation events are relevant in chromatin compaction and the initiation of cancer. It seems that methylation at certain locations of the DNA might be key to start chromatin compaction and gene silencing, but the location of this methylation sites is still unknown. In order to identify DNA methylation locations that could potentially be involved in chromatin compaction and gene silencing, this research focused on studying the effects of different DNA methylation patterns in the modulation of chromatin compaction.

Here, I engineered DNA sequences to include different DNA methylation patterns and test if their methylation status (methylated and unmethylated) had any influence in compactness of chromatin. The three methylation patterns studied consist of: 1) a stretch of methylation sites located at the middle of the DNA sequence ((CG)5) 2) methylation sites located at the major grooves ((CGX8)5,major) and minor grooves ((CGX8)5,minor) of the nucleosomal DNA. Using fluorescence spectroscopy techniques and other biophysical assays, I studied the effects of the methylation patterns on various properties of molecules representing three levels of chromatin organization: 1) Naked DNA, 2) Nucleosomes and 3) Nucleosome arrays. My results showed that compactness of chromatin-like molecules with (CG)5 and (CGX8)5,major patterns showed a dependence on their methylation status. Specifically, methylation of a stretch of (CG)5 decreased the relative compactness of nucleosomes and increased tetranucleosome compaction. The opposite effect was observed for nucleosomes and tetranucleosomes with (CGX8)5, major pattern. These findings confirm that the presence of methylation in certain locations within chromatin lead to distinctive effects on the compactness of chromatin-like molecules. Our results allowed us to identify two DNA methylation patterns that could potentially shed light onto DNA methylation locations that are more functionally significant for gene expression regulation. Although the biological relevance of these methylation locations is still to be determined, the results of this research are instrumental in understanding the mechanism of chromatin compaction by DNA methylation and could be applied in the identification of new and more accurate DNA methylation biomarkers for early detection of cancer.

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