Development of LC-MS method and myoblast differentiation model for studying the mechanism of DNA demethylation

Abstract

DNA methylation is an epigenetic modification important in many cellular processes such as maintenance of genome stability, establishment and maintenance of imprinting, transposon silencing, chromatin remodelling and control of gene expression. It is therefore important to understand how this modification is established and erased. We set out to develop a sensitive, liquid–chromatography mass–spectrometry method to measure global levels of DNA methylation (5mdC), as well as hydroxymethylation (5hmdC), a potential intermediate of DNA demethylation. With the new Agilent 6490 QQQ LC–MS we were able to detect as little as 50 amol of 5mdC and 5hmdC. We used this method to quantify levels of DNA methylation from DNA extracted from only 100 cells, allowing us to compare DNA methylation levels in early zygote development. Given that the evidence for DNA demethylation in early zygotes comes from methods using either antibody staining or bisulfite sequencing, this is the first direct demonstration that global DNA demethylation occurs in zygotes. Myoblast differentiation is a well-known model of DNA demethylation, where locus-specific DNA demethylation at the promoters and enhancer elements of myogenic regulatory factors, such as myogenin and MyoD, induce muscle specification and differentiation. It has been proposed, however, that the DNA demethylation process involves a large proportion of the genome and that it occurs in the absence of replication, indicating an active process. Therefore, in the second part of this work I set out to further investigate the global scale of epigenetic events associated with myoblast differentiation. Whilst some of the myoblast differentiation experiments showed a marked wave of DNA demethylation (up to 51%) others did not show any changes in DNA methylation level, showing that myoblast differentiation and DNA demethylation are not co-dependent. Addition of a DNA demethylating agent, 5-Aza-3’-deoxycytidine, to the growth medium of differentiating myoblast enhanced the differentiation process. On the other hand, inhibition of Poly (ADP- ribose) polymerase 1 activity, which has been shown to be mechanistically involved in DNA demethylation, inhibited the differentiation process. My work documents the kinetics of 5- v methylcytosine abundance during the myoblast differentiation together with the expression dynamics of factors previously linked to the DNA demethylation process.