Kinetics of Passive Demethylation

Abstract

DNA methylation is an epigenetic modification established during cellular differentiation, and when removed by either active or passive means, can substantially improve the efficiency by which induced pluripotent stem cells are created for the sake of regenerative medicine. During the ‘active’ removal of DNA methylation, the Ten-Eleven Translocation (TET) enzymes catalyse the oxidation of cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) and further derivatives. Unpublished work from the Hore laboratory and collaborators has shown that TET differentially targets particular CG-containing hexamer motifs for rapid demethylation but leaves others unaffected. Currently, it is unclear if ‘passive’ epigenetic memory loss, whereby demethylation results from the inhibition or inactivation of the DNA methylation maintenance machinery, also shows a preference for certain CG-containing hexamers. DNMT1 typically acts to maintain DNA methylation by catalysing the addition of methyl groups on hemimethylated DNA, enabling the transmission of methylation after DNA replication or repair. A cytosine-analogue called decitabine, which is also used in cancer treatment, can be utilised to block DNMT1 activity.

In this project, I used bisulfite sequencing data from decitabine-treated mouse embryonic stem cell samples to determine the kinetics of demethylation at all 256 CG-containing hexamers over a 48-hour period. A bioinformatic pipeline was implemented for the extraction and analysis of CG-containing hexamers from trimmed and mapped sequencing reads. Demethylation rates from these hexamers were examined both within and outside of CpG islands (CGI) and my analysis was performed in both absolute and relative terms according to similar experiments performed recently. In doing so, I found that once starting methylation levels and genomic location were taken into account, passive demethylation of DNA showed no preference to certain CG-containing hexamers, in stark contrast to that of active DNA demethylation.

In studying the kinetics of passive demethylation, I was able to answer a fundamental biological question regarding the mechanism by which passive demethylation occurs. However, in an applied sense, the value of this work will help to assess merits and risks of DNA demethylation strategies for the efficient creation of induced pluripotent stem cells that are safe for clinical use.