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Identification of an Enhancer of the Human Activating Protein-2 Gene That Contains a Critical Ets1 Binding Site

Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, Division of Endocrinology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039

Address all correspondence and requests for reprints to: You-Hong Cheng, Ph.D., Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. E-mail: cheny0{at}cchmc.org.

Deletion analysis studies of the 5'-flanking region of the activating protein-2 (AP-2) gene indicates that the proximal 152 bp are essential for minimal promoter activity and that a 140-bp fragment from nucleotides -1279 to -1139 acts as an enhancer of basal transcriptional activity. Ligation of the 140-bp fragment to a minimal AP-2 promoter or a heterologous simian virus 40 promoter luciferase reporter plasmid conferred enhancer activity in trophoblast cells. In deoxyribonuclease I footprint studies, nuclear extracts from trophoblast cells protected two regions of the 140-bp fragment, E2 and E3. Site-directed mutagenesis of an Ets1-binding site in E2 significantly inhibited AP-2 enhancer activity, whereas mutagenesis of two putative polyomavirus enhancer activator-3-binding sites on E2 and an insulin upstream factor 1-binding site in E3 had no effect on enhancer activity. Gel shift and supershift assays indicated that Ets1 binds to the Ets site in E2, and overexpression of Ets1 in transfection studies induced AP-2 promoter activity. As the transcription factor Ets1 is abundant in trophoblast cells, these investigations strongly suggest that AP-2 gene expression in the placenta is enhanced by a cis-acting element at nucleotides -1279 to -1139 that contains a critical Ets1-binding site.

This work was supported by NIH Grant HD-07447.

Abbreviations: AP-2, Activating protein-2; DNase I, deoxyribonuclease I; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCRH, human chorionic gonadotropin; IUF1, insulin upstream factor 1; Luc, luciferase; mtPEA3, mutant polyomavirus enhancer activator-3; nt, nucleotide; SV40, simian virus 40; TBST, Tris (hydroxymethyl) aminomethane-buffer saline containing 0.05% Tween 20; TK, thymidine kinase.

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