We report a single-cell bisulfite sequencing method (scBS-Seq) capable of accurately measuring DNA methylation at up to 48.4% of CpGs. We observed that ESCs grown in serum or 2i both display epigenetic heterogeneity, with “2i-like” cells present in serum cultures. In silico integration of 12 individual mouse oocyte datasets largely recapitulates the whole DNA methylome, making scBS-Seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

Imprinted genes in mammals are expressed from only one of the parental chromosomes, and are crucial for placental development and fetal growth. The insulin-like growth factor II gene (Igf2) is paternally expressed in the fetus and placenta. Here we show that deletion from the Igf2 gene of a transcript (P0) specifically expressed in the labyrinthine trophoblast of the placenta leads to reduced growth of the placenta, followed several days later by fetal growth restriction. The fetal to placental weight ratio is thus increased in the absence of the P0 transcript. We show that passive permeability for nutrients of the mutant placenta is decreased, but that secondary active placental amino acid transport is initially upregulated, compensating for the decrease in passive permeability. Later the compensation fails and fetal growth restriction ensues. Our study provides experimental evidence for imprinted gene action in the placenta that directly controls the supply of maternal nutrients to the fetus, and supports the genetic conflict theory of imprinting. We propose that the Igf2 gene, and perhaps other imprinted genes, control both the placental supply of, and the genetic demand for, maternal nutrients to the mammalian fetus.

Elucidating how and to what extent CpG islands (CGIs) are methylated in germ cells is essential to understand genomic imprinting and epigenetic reprogramming1-3. Here, we present the first integrated epigenomic analysis of mammalian oocytes, identifying over a thousand CGIs methylated in mature oocytes. We show that these CGIs depend on DNMT3A and DNMT3L4-5, but are not distinct at the sequence level, including in CpG periodicity6. They are preferentially located within active transcription units and are relatively depleted in H3K4me3, supporting a general transcription-dependent mechanism of methylation. Very few methylated CGIs are fully protected from post-fertilisation reprogramming but, surprisingly, the majority exhibits incomplete demethylation in E3.5 blastocysts. Our study shows that CGI methylation in gametes is not entirely related to genomic imprinting, but is a strong factor in determining methylation status in preimplantation embryos, suggesting a need to reassess mechanisms of post-fertilization demethylation.