An Apple a Day Keeps the DNA Methylation Away! Vitamin C and DNA Demethylation in Mouse ES Cells
DNA methylation is an epigenetic modification that is essential for normal development and also plays important roles in diverse cellular functions, such as genomic imprinting, X-chromosome inactivation, suppression of repetitive DNA elements, carcinogenesis, and other human diseases. In the last few years, scientists have focused on better understanding the process of active DNA demethylation in cells. Recent findings indicate that DNA demethylation is mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). Blaschke et al. found that vitamin C, which can be found in fruits such as apples and oranges, induces Tet-dependent DNA demethylation and a blastocyst-like state in mouse embryonic stem (ES) cells when present in cell culture media.
The scientists made an interesting discovery while culturing mouse embryonic stem (ES) cells in knockout serum replacement (KSR) conditions. The ES cells in this media showed a strong and reversible increase of expression of the germline gene Daz1. Previous publications have shown that the composition of the media can have an influence on ES cell heterogeneity, gene expression, and epigenetic patterns. Therefore, the scientists performed experiments to determine what component of the media caused the observed induction of the expression of Daz1, as well as to determine the underlying mechanism. A small molecule screen of the KSR media identified the antioxidant vitamin C as the component likely responsible for the increased Daz1 expression. It is important to note that mouse ES cells are usually cultured in vitamin C-free media. The scientists next tested different antioxidants (e.g. gluthatione or dithiothreitol) and found that they do not have the same effect as vitamin C, indicating the specificity of vitamin C in stimulating Daz1 expression.
It is known that vitamin C enhances the activity of some Fe(II) 2-oxogluterate dioxygenases, and therefore the authors postulated that vitamin C could promote the activity of Tet enzymes, which have this catalytic domain. The authors proposed that increased Tet activity could lead to increased DNA demethylation, and therefore induction of Daz1 expression. To see if vitamin C had an effect on the DNA methylation levels at specific regions in the genome, they investigated ES cells after 12 and 72 hours of vitamin C treatment by performing 5-hmC and 5-mC immunoprecipitation assays, followed by deep sequencing. The authors found that most of the methylated promoter regions showed increased levels of 5-hmC at 12h and they returned to normal levels by 72h. In contrast, 5-mC levels were decreased at both 12 and 72h after vitamin C treatment. Interestingly, there was a significant overlap in the promoters that have increased 5-hmC levels and those that show decreased 5-mC levels, in support of the hypothesis that oxidation of 5-mC to 5-hmC precedes DNA demethylation.
However, certain regions of the genome (some imprinted regions and A particle retroelements, for example) seem to be resistant to vitamin C-induced DNA demethylation. To see if the effect of vitamin C is really mediated by the Tet enzymes the authors investigated the 5-mC and 5-hmC changes in Tet1 and Tet2 double knockout ES cells after treatment. The scientists found no significant changes in these knockout cells, suggesting that vitamin C is a direct regulator of Tet activity. Furthermore, the researchers demonstrated that the DNA demethylation effect of vitamin C was significantly more pronounced in cultured ES compared to blastocysts.
Based on the results of this study, the authors concluded that vitamin C is a direct regulator of Tet activity and therefore has an impact of the level of DNA demethylation in ES cells. Will knowing about this report make you pay more attention the concentrations of vitamin C and other components in the media used in your experiments?
Blaschke K, Ebata KT, Karimi MM, Zepeda-Martínez JA, Goyal P, Mahapatra S, Tam A, Laird DJ, Hirst M, Rao A, Lorincz MC, & Ramalho-Santos M (2013). Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature PMID: 23812591