Whole-genome profiling of DNA methylation and hydroxymethylation identifies distinct regulatory programs among innate lymphocytes

Innate lymphocytes encompass a diverse array of phenotypic identities with specialized functions. DNA methylation and hydroxymethylation are essential for epigenetic fidelity and fate commitment. The landscapes of these modifications are unknown in innate lymphocytes. Here, we characterized the whole-genome distribution of methyl-CpG and 5-hydroxymethylcytosine in mouse ILC3, ILC2, and NK cells. We identified differentially methylated and hydroxymethylated DNA regions between ILC-NK subsets and correlated them with transcriptional signatures. We associated lineage-determining transcription factors with demethylation and demonstrated unique patterns of DNA methylation/hydroxymethylation in relationship to open chromatin regions, histone modifications, and transcription factor binding sites. We further discovered a novel association between hydroxymethylation and NK cell super-enhancers. Using mice lacking DNA hydroxymethylase TET2, we showed its requirement for optimal production of hallmark cytokines by ILC3 and IL-17A by inflammatory ILC2. These findings provide a powerful resource for studying innate lymphocyte epigenetic regulation and decode the regulatory logic governing their identity.