Weaning-Driven Gut Microbiome Shapes Intestinal Stem Cell Epigenetics to Train Immunological Memory (WGBS)

During weaning, the transition from milk to solid food rapidly expands and diversifies the gut microbiome, triggering the weaning reaction, a programmed immune response critical for long-lasting mucosal immunity. We characterized how these microbiome changes from weaning through young adulthood in mice shape the DNA methylome and transcriptome of intestinal stem cells (ISCs). We identified a weaning-induced IFN-g–TET3 axis as a key driver of MHC class II enhancer-linked epigenetic reprogramming, creating a transcriptional memory that persists through differentiation into adulthood. Early-life disruption of this axis, via IFN-g blockade or low-dose penicillin, impaired microbiota-mediated epigenetic control and mucosal immunity. Weaning-associated microbiome shifts enriched metabolites such as a-ketoglutarate, Fe2+, and methionine g-lyase, promoting TET-dependent DNA demethylation. These findings reveal how early-life events imprint lifelong epigenetic regulation of mucosal immunity, highlighting epithelial cells as innate immune sentinels that acquire trained transcriptional memory in response to microbial cues. Overall design: Genome-wide DNA methylation profiling by WGBS was performed in mice colonic stem cells and differentiated cells at 3 weeks and 15 weeks of age.