DOT1L is essential for corneal epithelial identity and homeostasis

The corneal epithelium is maintained by limbal stem cells (LSCs) that differentiate into mature epithelial cells to ensure transparency and barrier function. Epigenetic regulation is critical for corneal homeostasis, yet the role of histone methyltransferase DOT1L, the sole enzyme catalyzing H3K79 methylation, remains unexplored. Here, we show that DOT1L is indispensable for corneal epithelial differentiation, postnatal development, and wound healing. Using conditional knockout mice (Dot1lflox/flox; K14-Cre), we find that DOT1L deficiency causes progressive corneal opacification, squamous metaplasia, and pathological angiogenesis. Immunofluorescence and histochemical analyses reveal that DOT1L deficiency impedes corneal epithelial differentiation, consequently inhibiting both postnatal corneal development and wound-induced corneal repair. Single-cell RNA sequencing of WT and Dot1l-KO corneas demonstrates that DOT1L loss disrupts the LSC-to-superficial-cell differentiation trajectory, and identifies Pax6, the master regulator of corneal epithelial identity, as one of the differentially expressed genes. Mechanistically, DOT1L directly deposits H3K79me2 at the Pax6 promoter to activate its transcription. Concurrently, DOT1L deficiency represses interferon signaling genes, coinciding with pathological angiogenesis. Collectively, our findings establish DOT1L as a critical guardian of corneal epithelial identity through regulation of epithelial differentiation, positioning DOT1L as a potential therapeutic target for corneal disorders.