During the mammalian life cycle, transitions in developmental potency and cell fate are closely associated with extensive reprogramming of the epigenome. One such event occurs shortly after fertilisation when 5-methylcytosine (5mC), a well-established epigenetic mark, is rapidly removed from the paternal genome as part of global reorganisation thought to be important for the totipotency of the newly formed embryo. Strikingly, the later phase of this demethylation coincides with an accumulation of 5-hydroxymethylcytosine (5hmC), a base generated from oxidation of 5mC by TET enzymes that can provide a pathway to unmodified cytosine.
To probe the role of 5hmC in zygotic demethylation, we generated C57BL/6 mice carrying a floxed allele of Tet3, the TET family member responsible for oxidation in the zygote; expression of Cre under the Zp3 promoter results in oocytes lacking TET3 oxidase activity. Immunofluorescence revealed that upon fertilisation by wild-type sperm, 5hmC failed to accumulate and demethylation was impaired in the male pronucleus, consistent with other reports that the oxidative pathway is required for normal demethylation1,2. Despite this defective reprogramming, embryos developed normally and were healthy and fertile after birth. This contrasts with the compromised embryonic development observed in a similar study using mixed C57BL/6J-129Sv mice2, indicating that genetic background may be an important modifier of the requirement for full reprogramming.
To pinpoint which sequences are demethylated by oxidation, we optimised a whole-genome bisulfite sequencing technique for low cell numbers (<300) and applied it to TET3-deficient zygotes. In addition to providing the first genome-wide methylation profile of zygotes, this analysis confirmed a global trend of increased methylation resulting from loss of TET3 and identified specific loci affected by the lack of oxidative demethylation. These molecular insights advance our understanding of the complex demethylation network operating in the zygote and provide a resource for assessing the impact of epigenetic reprogramming on development.