Vitamin C is a direct regulator of Tet enzyme activity and DNA methylation fidelity in mouse ES cells; addition of vitamin C promotes Tet activity, increases 5-hydroxymethlycytosine (5hmC) and DNA demethylation of many gene promoters, upregulates demethylated germline genes, and induces a state that more closely approximates that of the inner cell mass of the blastocyst. The Tet enzymes regulate DNA methylation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized variants. Here vitamin C, a cofactor of enzymes of the same family as the Tets, is shown to be a direct regulator of Tet activity in mouse embryonic stem cells, which are typically cultured in the absence of vitamin C. Addition of vitamin C to the culture medium leads to increased 5hmC content and the demethylation of numerous gene promoters. The remodelled DNA methylation and gene expression patterns resemble the DNA demethylation that occurs in the inner cell mass of the early embryo. DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons1. Global DNA demethylation occurs in the early embryo and the germ line2,3, and may be mediated by Tet (ten eleven translocation) enzymes4,5,6, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC)7. Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells8,9,10,11,12, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(ii) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo2,13, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has enabled genome-wide epigenetic profiling of numerous cell lines and tissue types. A major limitation of ChIP-seq, however, is the large number of cells required to generate high-quality data sets, precluding the study of rare cell populations. Here, we present an ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) and sequencing method to generate genome-wide histone mark profiles with high resolution from as few as 10³ cells. We demonstrate that ULI-NChIP-seq generates high-quality maps of covalent histone marks from 10³ to 10⁶ embryonic stem cells. Subsequently, we show that ULI-NChIP-seq H3K27me3 profiles generated from E13.5 primordial germ cells isolated from single male and female embryos show high similarity to recent data sets generated using 50–180 × more material. Finally, we identify sexually dimorphic H3K27me3 enrichment at specific genic promoters, thereby illustrating the utility of this method for generating high-quality and -complexity libraries from rare cell populations.