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

Innate lymphocytes encompass a diverse array of phenotypic identities with specialized effector functions. DNA methylation and hydroxymethylation are essential mechanisms 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 (5hmC) in mouse ILC3, ILC2, and NK cells. We identified differentially methylated and hydroxymethylated DNA regions between ILC-NK subsets and correlated them with signature transcriptional modules. 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 also discover a novel association between 5hmC and NK cell super-enhancers. Using Tet2-deficient mice, we showed that the DNA hydroxymethylase Tet2 is required for optimal production of hallmark cytokines by ILC3 and production of IL-17A by inflammatory ILC2. These findings provide a powerful resource for studying immune epigenetic regulation and further decode the regulatory logic governing innate lymphocyte identity. Splenic NK cells, intestinal ILC2, and intestinal ILC3 were sorted from mice and MeDIP-seq, MRE-seq, and hmC-seal libraries were generated. Two biological replicates were used for each cell type.

固有淋巴细胞（Innate Lymphocytes）涵盖了一系列多样化的表型特征，并具备特化的效应功能。DNA甲基化与羟甲基化是维持表观遗传稳定性与细胞命运决定的核心机制，但目前针对固有淋巴细胞的此类修饰图谱仍未被完全阐明。本研究对小鼠ILC3、ILC2以及NK细胞中全基因组范围内的甲基化CpG位点与5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）的分布特征进行了系统解析。研究团队鉴定出了ILC-NK细胞亚群之间的差异甲基化区域与差异羟甲基化区域，并将这些区域与特征性转录模块进行了关联分析。本研究将谱系决定性转录因子与DNA去甲基化过程建立关联，并揭示了DNA甲基化/羟甲基化与开放染色质区域、组蛋白修饰以及转录因子结合位点之间的独特关联模式。此外，本研究还发现了5hmC与NK细胞超级增强子之间的全新关联。借助Tet2基因敲除小鼠模型，本研究证实DNA羟甲基化酶Tet2对于ILC3分泌标志性细胞因子以及炎症性ILC2产生IL-17A均具有关键调控作用。上述研究成果为免疫表观遗传调控研究提供了宝贵的资源，并进一步解析了调控固有淋巴细胞身份特征的表观遗传逻辑。本研究从小鼠体内分选了脾脏NK细胞、肠道ILC2以及肠道ILC3，并构建了MeDIP-seq、MRE-seq以及hmC-seal测序文库，每种细胞类型均设置两个生物学重复。