Suppression of Krüppel-like factor 5 basal expression by CREB1 binding to far distal element

Abstract

BACKGROUND: Krüppel-like factor 5 (KLF5) is a transcription factor regulating the proliferation and differentiation of epithelial cells, and its uncontrolled expression is closely associated with carcinoma progression. Sp3 binding to the minimal essential region (MER) of KLF5 gene is critical for KLF5 basal expression, but the expression control mechanism is unknown. OBJECTIVE: This study aimed to identify a regulatory region for KLF5 basal expression and the binding protein in carcinoma cells by analyzing the promoter upstream region. METHODS: Reporter assays determined the silencer region. The protein binding to the region was identified by database analysis and ChIP assay. The protein mediating the interaction between the region and the MER was confirmed through chromosome conformation capture (3 C) on ChIP assay. The effects of the protein on KLF5 expression were analyzed using qRT-PCR and western blot. RESULTS: Reporter assay localized the 425-region from upstream KLF5 gene as the silencer. Database analysis and ChIP assay found CREB1 binding to the 425-region. CREB1 siRNA or mutation of CREB1-binding site in the 425-region increased luciferase activities and decreased the binding to 425-region. 3 C on ChIP assay showed that CREB1 mediated interaction of the 425-region and the MER. CREB1 overexpression decreased endogenous KLF5 expression and luciferase activity. CONCLUSIONS: The 425-region is the silencer of KLF5 basal expression, and CREB1 binding suppresses the expression.