Identification of a Distal Enhancer That Regulates TGF-β-Induced SNAI1 Expression

Snail is a zinc finger transcription factor encoded by the SNAI1 gene and triggers a cellular process termed epithelial-mesenchymal transition (EMT) upon its increased expression and/or functional activation. Snail expression and activity are regulated by various extracellular stimuli, including cytokines and environmental factors. Transforming growth factor-β (TGF-β) is a Snail inducer that functions via Smad3-mediated transcriptional activation. In the present study, we identified a distal enhancer that modulates TGF-β-induced SNAI1 expression. ChIP-seq and Hi-C analyses showed that the enhancer is located 46 kb downstream of the SNAI1 gene; in TGF-β-stimulated cells, it associates with Smad3 and interacts with the SNAI1 proximal promoter. Inhibiting the activity of the enhancer using CRISPRi attenuated TGF-β-induced SNAI1 expression, stress fiber formation, and cell motility enhancement, suggesting that the enhancer mediates TGF-β-induced EMT. The enhancer contains a Smad-binding CAGA motif and an activator protein-1 (AP-1) binding motif that function in transcriptional activation. Ras-responsive element binding protein 1 (RREB1), a transcription factor required for TGF-β-induced Snail expression, regulated the basal activity of the enhancer but not its inducibility by TGF-β. In contrast to the enhancer, the association of Smad3 with the proximal promoter was not evident. These findings suggest that the proximal promoter and the distal enhancer respond to distinct signaling cues, integrate them, and cooperatively function to drive SNAI1 expression.