Single-cell 5-hydroxymethylcytosine landscapes of mouse early embryos at single-base resolution

DNA methylation and hydroxymethylation are extensively reprogrammed during mammalian early embryogenesis, and studying their regulatory functions requires comprehensive DNA hydroxymethylation maps at base resolution. Here, we develop single-cell 5-hydroxymethylcytosine (5hmC) chemical-assisted C-to-T conversion-enabled sequencing (schmC-CATCH), a method leveraging selective 5hmC labeling for a quantitative, base-resolution, genome-wide landscape of the DNA hydroxymethylome in mouse gametes and preimplantation embryos spanning from the zygote to blastocyst stage. We revealed that, in addition to late zygotic stages, onset of ten-eleven translocation (TET)-mediated DNA hydroxymethylation initiates immediately after fertilization and is characterized by the distinct 5hmC patterns on the parental genomes shaped by TET3 demethylase. We identified persistent clusters of 5hmC hotspots throughout early embryonic stages, which are highly associated with young retroelements. 5hmC is also associated with different regulatory elements, indicating a potential regulatory function during early embryogenesis. Collectively, our work elucidates the dynamics of active DNA demethylation during mouse preimplantation development and provides a valuable resource for functional studies of epigenetic reprogramming in early embryos.