The CXXC1-IGFBP6 Axis Maintains Corneal Epithelial Differentiation via H3K4me3-Dependent Transcriptional Activation

Purpose: To define the role and mechanisms of the epigenetic regulator CXXC1 in corneal epithelial cells (CECs) differentiation. Methods: Chromatin remodeling-related gene ontology (GO) modules were integrated with RNA sequencing (RNA-seq) to identify CXXC finger protein 1 (CXXC1) as a candidate epigenetic regulator of CECs differentiation. Loss-of-function studies were performed in limbal stem/progenitor cells (LSCs), followed by air-liquid interface-induced differentiation into CECs. Effects of CXXC1 depletion were assessed by RNA-seq, quantitative real-time PCR, and immunofluorescence. Chromatin immunoprecipitation sequencing (ChIP-seq) for CXXC1 and H3K4me3 was combined with transcriptomics to identify insulin-like growth factor binding protein 6 (IGFBP6) as a direct target. The role and pathological relevance of the CXXC1-IGFBP6 axis were evaluated by IGFBP6 knockdown and in human corneal ulcer tissues. Results: CXXC1 was expressed throughout the corneal epithelium and was markedly upregulated during LSC-to-CEC differentiation. CXXC1 depletion impaired differentiation and reduced expression of CECs markers and key corneal epithelial regulators. ChIP-seq revealed CXXC1 binding at the IGFBP6 locus, coinciding with H3K4me3 enrichment, whereas CXXC1 knockdown reduced both H3K4me3 enrichment and IGFBP6 expression. IGFBP6 knockdown phenocopied CXXC1 loss. Depletion of either gene activated inflammatory, angiogenic, and hypoxia-related gene programs, suggesting abnormal corneal epithelial differentiation and pathological changes. Both proteins were markedly reduced in human corneal ulcer lesions. Conclusions: CXXC1 maintains CECs differentiation through H3K4me3-dependent activation of IGFBP6 and other epithelial regulators. The CXXC1-IGFBP6 axis is associated with pathological changes in corneal ulcer lesions.