Identification of an enhancer of the human activating protein-2alpha gene that contains a critical Ets1 binding site.
Deletion analysis studies of the 5'-flanking region of the activating protein-2alpha (AP-2alpha) 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-2alpha 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-2alpha 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-2alpha promoter activity. As the transcription factor Ets1 is abundant in trophoblast cells, these investigations strongly suggest that AP-2alpha gene expression in the placenta is enhanced by a cis-acting element at nucleotides -1279 to -1139 that contains a critical Ets1-binding site.
Cheng
You-Hong
YH
Cincinnati Children's Hospital Medical Center and Department of Pediatrics, Division of Endocrinology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039, USA. cheny0@cchmc.org
Handwerger
Stuart
S
D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005260:Female; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D014327:Trophoblasts / Q000166:cytology / Q000502:physiology
D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; C493673:TFAP2A protein, human; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D003850:Deoxyribonuclease I