10446912

pubmed:Citation

8

The ovine P45 side chain cleavage (CYP11A1) enzyme gene, which catalyzes the initial enzymatic step in steroid hormone biosynthesis is transcriptionally regulated in cultured steroidogenic human trophoblastic JEG-3 cells. The ovine CYP11A1 promoter contains two GC-rich footprinted regions referred to as ovine footprints 5 (OF5) and OF3, which are well conserved among the CYP11A1 promoters of different species. These GC-rich sequences resemble activator protein-2 (AP-2)/Sp1 binding sites and were previously implicated in basal and cAMP-regulated activity of the bovine and ovine CYP11A1 promoters. In the current studies, AP-2 induced the ovine CYP11A1 promoter 4.5-fold in JEG-3 cells with full induction requiring the previously defined cAMP-responsive elements. Point mutation of OF3 abolished induction by AP-2, and OF3 was sufficient for induction by AP-2 when linked to a heterologous promoter. AP-2 induction of the CYP11A1 promoter required the basic region (N165-N278) and the carboxy terminus of AP-2 (N413-N437). In the course of investigating the mechanisms by which OF5 and OF3 regulated CYP11A1 transcription, we found that OF5 and OF3 bound Sp1 and Sp3 in JEG-3 cells. AP-2 did not bind OF5 or OF3 directly but rather formed a multiprotein complex with Sp1 in JEG-3 cells. AP-2 associated directly with Sp1 in vitro requiring the AP-2 basic region and the Sp1 carboxy terminus. AP-2 induced Sp1/Sp3 activity independently of AP-2 binding to DNA using a GAL4 paradigm. The Sp1 and Sp3 transactivation domains were linked to the DNA-binding domain of GAL4, and their activity was assessed using a luciferase reporter gene containing only the GAL4 DNA-binding sites linked to the minimal TATA site. AP-2 induced Sp1/ Sp3-GAL4 activity 3- to 4-fold, requiring both the amino and extreme carboxy terminus of AP-2. We conclude that AP-2 can bind to and stimulate Sp1 activity and induces the ovine CYP11A1 promoter through conserved Sp1/Sp3-binding sites in JEG-3 cells. The induction of Sp1 activity by AP-2 may contribute to the induction of other genes that bind Sp1.

eng

IM

MEDLINE

0888-8809

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NLM

1402-16

pubmed-meshheading:10446912-Animals, pubmed-meshheading:10446912-Binding Sites, pubmed-meshheading:10446912-Cell Line, pubmed-meshheading:10446912-Cercopithecus aethiops, pubmed-meshheading:10446912-Cholesterol Side-Chain Cleavage Enzyme, pubmed-meshheading:10446912-Choriocarcinoma, pubmed-meshheading:10446912-DNA, pubmed-meshheading:10446912-DNA-Binding Proteins, pubmed-meshheading:10446912-Gene Deletion, pubmed-meshheading:10446912-Humans, pubmed-meshheading:10446912-Kidney, pubmed-meshheading:10446912-Point Mutation, pubmed-meshheading:10446912-Promoter Regions, Genetic, pubmed-meshheading:10446912-Regulatory Sequences, Nucleic Acid, pubmed-meshheading:10446912-Sheep, pubmed-meshheading:10446912-Sp1 Transcription Factor, pubmed-meshheading:10446912-Structure-Activity Relationship, pubmed-meshheading:10446912-Transcription, Genetic, pubmed-meshheading:10446912-Transcription Factor AP-2, pubmed-meshheading:10446912-Transcription Factors, pubmed-meshheading:10446912-Tumor Cells, Cultured

Activator protein-2 mediates transcriptional activation of the CYP11A1 gene by interaction with Sp1 rather than binding to DNA.

Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't