Novel Approaches in DNA Methylation Analysis: Sequencing Strategy Development and Application to Cancer Metastasis Studies

The study of epigenetics, particularly DNA methylation, provides crucial insights into gene regulation and its aberrations in diseases such as cancer. This dissertation is anchored in the exploration of DNA methylation, a fundamental epigenetic modification critically involved in cellular differentiation, developmental processes, and tumorigenesis. To advance the exploration of methylation landscapes, we have developed a novel enzymatic-based, bisulfite-free sequencing strategy, TT-5mC-seq. This method achieves single-base resolution in the detection of 5-methylcytosine (5mC) modifications, markedly reducing background noise without causing damage to the DNA. On the other hand, we have characterized the patterns of 5-hydroxymethylcytosine (5hmC) across various human tissues, both normal and cancerous, revealing the subtle yet consequential shifts in the epigenetics associated with disease states. This research provides not only new methodologies for probing DNA methylation but also enhances our understanding of 5hmC's potential as a biomarker for cancer metastasis.