CHIR99021 enhances Klf4 Expression through β-Catenin Signaling and miR-7a Regulation in J1 Mouse Embryonic Stem Cells

Understanding the mechanisms that regulate pluripotency of embryonic stem cells (ESCs) is important to ensure their safe clinical use. CHIR99021 (CHIR)-induced activation of Wnt/beta-catenin signaling promotes self-renewal in mouse ESCs (mESCs). beta-catenin functions individually or cooperates with transcription factors to activate stemness factors such as cMyc, Esrrb, Pou5f1, and Nanog. However the relationship between the core pluripotent factor, Kruppel-like factor 4 (also known as GKLF or EZF) and Wnt/beta-catenin signaling, remains ambiguous in J1 mESCs. DNA microarray analysis revealed that CHIR-treatment promoted pluripotency-maintaining transcription factors and repressed germ layer specification markers. CHIR also promoted genes related to the development of extracellular regions and the plasma membrane to maintain pluripotency of J1 mESCs. Among the CHIR-regulated genes, Klf4 has not been reported previously. We identified a novel cis element in the Klf4 gene that was activated by beta-catenin in J1 mESCs. We determined that beta-catenin interacted with this cis element, identifying Klf4 as a beta-catenin target gene in this context. Moreover, several microRNAs that targeted the 3'-UTR of Klf4 mRNA were identified, with miR-7a being down-regulated by CHIR in a beta-catenin-independent manner in J1 mESCs. These data collectively suggest that CHIR enhances Klf4 expression by repressing miR-7a expression or canonical Wnt pathway activation.