YTHDF2-KIF26B-Wnt signaling forms a positive-feedback regulatory loop to maintain ISC stemness

m6A modification plays a regulatory role in the stemness of small intestinal epithelial stem cells (ISCs), which is crucial for the renewal of intestinal epithelium and the maintenance of homeostasis. Although existing research has demonstrated that METTL3, as an m6A ‘writer’, is a crucial protein in the regulation of the stemness of ISCs, the mechanism by which the function of downstream ‘reader’ proteins is regulated has not been fully elucidated. In this study, we found that the knockout of Ythdf2 could significantly down-regulate the expression of the stemness marker gene Lgr5, suggesting that this ‘reader’ protein plays an important role in the regulation of the stemness of ISCs. Upon exploring the specific downstream mechanisms, we discovered that YTHDF2 promoted the Wnt signaling pathway by regulating the expression of KIF26B. This helps maintain the expression of Lgr5 in stem cells, thus preserving the stemness of these cells. Meanwhile, the expression of YTHDF2 itself is also regulated by the Wnt signaling pathway. Overall, YTHDF2 forms a positive-feedback regulatory loop centered around the Wnt signaling pathway. This loop ensures that ISCs remain in a stem-cell state, enabling the intestinal epithelium to function stably and properly. Cultivate the small intestinal epithelial organoids of Villin-CreERT2; Lgr5-EGFP-IRES-CreERT2; Ythdf2fl/fl mice in vitro. The Lgr5-GFP+ cells are sorted on the 4th or 6th day after the treatment with 0.5 μM EtOH/4-OHT. Then their RNA was collected and performed RNA sequencing.