Methylated DNA Immunoprecipitation Sequencing
DNA-methylation is an enzymatically (DNA methyltransferase (DNMT) enzymes) conversion of a cytosine residue into a 5-methylctosine (5mC), which use S-adenosyl methionine (SAM) as the methyl donor. It is a type of epigenetic modification resulting in gene silencing. In addition to DNA methylation, gene expression can be regulated by other processes, such as: RNA turnover, mRNA processing and transcription rate. The 5mC can be enzymatically (ten-eleven-translocation (TET) enzymes) converted into 5-hydroxymethylcytosine (5hmC). This 5mC to 5hmC conversion may facilitate passive DNA demethylation by excluding maintenance of DNA methylation during cell division by DNMT1, which recognizes 5hmC poorly, suggesting that the presence of 5hmC will stop the gene silencing effect of 5mC. Recently, it has been shown that not only nuclear DNA (nDNA), but also mitochondrial DNA (mtDNA) may be subjected to epigenetic modifications (e.g. methylation). These mtDNA epigenetic modifications can modulate nDNA and nDNA epigenetic modifications may affect mtDNA.
It has been shown that oxidative stress plays a role in the Cà5mC and 5mCà5hmC conversion by increasing the DNMT and TET enzymes in nDNA. This mechanism is probably also involved in mtDNA. Furthermore it has been shown that oxidative stress plays a role in liver-toxicity. Therefore, the aim of this PhD project is to investigate how changes in patterns of 5mC and 5hmC, in nDNA and mtDNA, caused by oxidative stress are involved in the mechanisms of liver-toxicity. Our research questions are:
1. Does an oxidative stressor make normal liver cells more prone to (liver-)toxicity through hypo-methylation and-or hyper-hydroxymethylation? and,
2. Suppose that the mtDNA methylation levels change, what is than the effect on gene expression and DNA methylation in the nucleus and mitochondria?