We demonstrated that ETV4 is a transcriptional activator of the NANOG gene in human embryonic carcinoma NCCIT cells. The endogenous expression of NANOG and ETV4 in naive cells was significantly down-regulated upon differentiation and by shRNA-mediated knockdown of ETV4. NANOG transcription was significantly upregulated by ETV4 overexpression. A putative ETS binding site (EBS) is present in the region (-285 to-138) of the proximal promoter. Site-directed mutagenesis of the putative EBS ((-196)AGGATT-(191)) abolished NANOG promoter activity and ETV4 interacted with this putative EBS both in vivo and in vitro. Our data provide the molecular details of ETV4-mediated NANOG gene expression. (C) 2017 Elsevier Inc. All rights reserved.