EGR-1 is an active transcription factor in TGF-β2-mediated small intestinal cell differentiation

Human milk contains growth factors that maintain intestinal mucosal homeostasis, but the molecular mechanisms behind how these growth factors regulate gene transcription are largely unknown. In this study, IEC-6 (rat intestinal epithelial cells) cells were used as a model to study cell differentiation mediated by transforming growth factor-β2 (TGF-β2), the most abundant growth factor in human milk. We focused on the transcription factor early growth response-1 (EGR-1), as we found a robust and rapid response in our initial transcription factor screen. Immunoblotting and immunofluorescent assays confirmed the phenotype change upon TGF-β2 treatment and EGR-1 stimulation in the nucleus, with maximum expression occurring at 1 h. Chromatin immunoprecipitation sequencing was performed to map genome-wide EGR-1 binding sites on more than 1800 genes, widely involved in processes such as gene expression, transcription, membrane invagination and metabolism. In particular, more than 15 Wnt signaling pathway genes have EGR-1 binding sites; among them, Axin1 was the limiting factor, ensuring proper β-catenin accumulation in the cytoplasm. We further used chromatin immunoprecipitation quantitative PCR to validate that EGR-1 binds to the region of −636/−454 bp and −454/−200 bp of the Axin1 promoter and functionally activates gene expression. The effect of TGF-β2 on maintaining small intestinal cell homeostasis was partially explained by Axin1 activation through EGR-1.