title: Activator protein-2 mediates transcriptional activation of the CYP11A1 gene by interaction with Sp1 rather than binding to DNA.
abstract: 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.
authors.affiliation: The Albert Einstein Cancer Center, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: P
authors.initials: P
authors.lastname: Pena
authors.forename: A T
authors.initials: AT
authors.lastname: Reutens
authors.forename: C
authors.initials: C
authors.lastname: Albanese
authors.forename: M
authors.initials: M
authors.lastname: D'Amico
authors.forename: G
authors.initials: G
authors.lastname: Watanabe
authors.forename: A
authors.initials: A
authors.lastname: Donner
authors.forename: I W
authors.initials: IW
authors.lastname: Shu
authors.forename: T
authors.initials: T
authors.lastname: Williams
authors.forename: R G
authors.initials: RG
authors.lastname: Pestell
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D002822:Choriocarcinoma; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D017353:Gene Deletion; D006801:Humans; D007668:Kidney; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012756:Sheep; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013329:Structure-Activity Relationship; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004247:DNA; D002786:Cholesterol Side-Chain Cleavage Enzyme