Abstract Background: Colorectal cancer (CRC) arises from both genetic and epigenetic alterations. Epidemiological and animal studies suggest early-life exposures, including gut microbiota changes during weaning, can influence long-term CRC risk. However, the mechanisms linking early microbial interactions to durable disease protection remain unclear. We hypothesize that early microbial exposure epigenetically programs long-lived intestinal stem cells (ISCs), shaping immune regulation and disease susceptibility. Methods: Lgr5-GFP reporter mice were used to isolate colonic ISCs and intestinal epithelial cells (IECs) under specific-pathogen-free (SPF) and germ-free (GF) conditions. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing were performed to assess microbiota-associated transcriptional changes mediated by DNA methylation from weaning into adulthood. Gut microbe transplant (GMT) experiments defined the critical developmental window during which ISC epigenetics are most sensitive to microbial influences. A maternal low-dose penicillin (LDP) model combined with shotgun metagenomics identified specific bacterial taxa contributing to epigenetic effects. Dextran sulfate sodium (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced CRC models were used to evaluate long-term consequences. Results: We identified 683 differentially methylated regions (DMRs) that were persistently associated with the microbiome in both adult ISCs and IECs. Among these, 51% were located in enhancers, and the majority DMRs (79%) exhibited loss of methylation under SPF conditions. The hypomethylated genes were enriched for immune and host defense functions, including MHC class II genes (Cd74, H2-Aa, H2-Eb1, and Ciita). Loss of methylation occurred post-weaning and correlated with increased gene expression. GMT experiments demonstrated the post-weaning as the optimal window to restore methylation patterns, compared to adolescence or adulthood. Mechanistically, a transient IFN-γ burst during weaning drove epigenetic reprogramming via the IFN-γ-STAT3-TET3 axis. In addition, microbiota-derived metabolites, including short-chain fatty acids (SCFAs), α-ketoglutarate (α-KG), and methionine-γ-lyase-dependent products, reinforced the establishment of proper methylation patterns. Early-life LDP exposure reduced Gram-positive bacterial abundance, altered IFN-γ expression, disrupted MHC-II epigenetics, and increased susceptibility to colitis and CRC. Conclusion: Early-life microbial and immune signals establish durable epigenetic programs that protect against CRC. Timed microbial interventions may provide long-lasting protection against adult-onset diseases. Citation Format: Li Yang, Shirui Zhou, Xiaomin Chen, Fabiola Gutierrez, Stephanie Fowler, Lanjing Zhang, Julia M. Salamat, Karen Riggins, Jiejun Shi, Lanlan Shen. Early-life gut microbiota programs intestinal stem cell epigenetics to protect against colon cancer later in life abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1955.
Yang et al. (Fri,) studied this question.