Epigenetic imprinting in innate lymphoid cell precursors directs the lineage segregation of innate lymphoid cells.

Innate lymphoid cells (ILCs) are essential for mucosal homeostasis, but the epigenetic regulation of their lineage segregation remains elusive. Here we simultaneously profiled the single-cell DNA methylome, chromatin accessibility and transcriptome of ILC subsets and ILC precursors (ILCPs) and found that ILCPs could be divided into two subgroups (ILCP1 and ILCP2). ILCP2s had highly heterogeneous DNA methylation profiles and could be divided into three groups according to their DNA methylation characteristics, which matched those of ILC subsets. We identified the signature methylation regions (SMRs) of each ILC subset and traced the DNA methylation imprinting during ILCP differentiation. ILCP2s with hypomethylated SMRs characteristic of ILC subsets differentiated into those subsets. DNA methylation editing of SMRs suppressed ILC lineage segregation, while deletion of Dnmt1 in ILCPs abrogated the heterogeneous distribution of SMRs and resulted in ILC differentiation defects. These findings provide evidence that epigenetic imprinting determines lineage segregation during immune cell development.