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Project 4: ROS, DNA methylation and cardiac fibrosis

PI Göttingen: E.M. Zeisberg; PI London: K. Otsu; PhD: S.K. Rath - finished PhD

Scientific background and preliminary results

While accumulation of reactive oxygen species and ensuing oxidative damage have been unequivocally established as causal contributor to cardiovascular morbidity and mortality, underlying mechanisms are diverse and in their complexity still incompletely understood. This project aims to gain mechanistic insights into the mechanisms underlying the accumulation of aberrant DNA methylation patterns in response to oxidative stress. Specifically, this project will explore, how DNA oxidation interferes with the endogenous pathways which control methylation of CpG island promoter regions and how ensuing aberrant DNA methylation contributes to endothelial-mesenchymal transition (EndMT) and ultimately to cardiac fibrosis. Background: Our group and others previously discovered that EndMT – the acquisition of mesenchymal features by endothelial cells – contributes causally to cardiac fibrosis. We further discovered that such EndMT is dependent of aberrant CpG promoter methylation of select genes, including RASAL1. Mechanisms which are responsible for aberrant methylation status of specific genes are not yet understood in any biological system. However, our group established that in context of EndMT methylation status of select genes is determined by the interplay of the methylating enzyme DNA methyltransferase 1 (Dnmt1, which catalyzed addition of a methyl-group to the C5-postion of cytosine) and the ten eleven translocation-3 (Tet3) zinc finger protein (which mediates oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and also of thymidine to 5-hydroxymethyluracil), ultimately leading to base-excision repair (BER) and replacement by naked bases. While Tet3-mediated DNA oxidation is tightly regulated and depends on Tet3-recuitment to specific CXXC motifs in proximity to methylated CpGs, reactive oxygen species (ROS) can equally oxidase DNA – albeit in unspecific manner. While in carcinogenesis ROS-induced DNA oxidation is a well-established cause of cancer, the role of DNA-oxidation in response to oxidative stress has not been explored in cardiovascular disease. Based on our previous data and existing literature this project is based on the central hypothesis that uncontrolled DNA oxidation in response to oxidative stress interferes with physiological DNA-methylation mechanisms, ultimately contributing to aberrant DNA-methylation patterns and fibrosis.

Hypotheses of the PhD project

In the project the doctoral researcher will test the hypotheses that

1. ROS-induced DNA oxidation enhances Dnmt1-catalyzed DNA methylation, and inhibits Tet3-dependent hydroxymethylation and base-excision repair, stably methylating select genes including RASAL1.

2. Oxidative stress associated with diabetes mellitus and/or chronic kidney failure causally augments EndMT and cardiac fibrosis by ROS-induced changes in DNA methylation patterns.