title: Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter.
abstract: This study was designed to explore the possible existence and location of estrogen response elements (EREs) in the human insulin receptor (hIR) gene promoter. Transfections of U-937 cells with the reported plasmids phIR(-1819)-GL2, phIR(-1473)-GL2, and phIR(-876)-GL2, that contain the -1819 to -271 bp fragment of the hIR promoter (wild-type promoter) and progressive 5' deletions of this promoter, revealed that while the activity of the wild-type promoter, was repressed 36% by treatment with 17beta-estradiol (E(2)), the activities of progressive 5' deletions of this promoter were reduced by 26% and by 0%, by this hormone. This suggests that E(2) needs the wild-type promoter for full transcriptional repression of this gene and it also suggests the presence of putative EREs in the region between -1819/-877 bp of this promoter. To identify these EREs we performed a computer search, using the SEQFIND programme developed in our laboratory, by homology with the consensus vit-ERE (5'GGTCAnnnTGACC3') of the Xenopus vitellogenin A(2) gene promoter. The results of our search indicated no sequence identical to this consensus ERE, and neither was any sequence found to show 9 or 8 of the 10 bases of this consensus in this promoter. Nevertheless, a putative hIR ERE1 (5'AGTGAaacTGGCC3') showing 7 bases of the consensus vit-ERE, and 10 bases of the optimal binding sequence ERE (5'CA/GGGTCAnnnTGACCT/CG3'), was identified between -1430/-1418bp of the hIR promoter. An AP-1-like site was covering the 3' half-element of this ERE; another AP-1-like site was overlapping the first AP-1-like site, and finally a third AP-1-like site was located beside to the 5' half-element. In addition, another putative hIR ERE2 (5'GCTCCtagCAAAC3') showing 5 bases of the consensus vit-ERE, and 9 bases of the optimal binding sequence ERE, was located upstream of the hIR promoter, between -1567/-1555 bp. An AP-1-like site was located downstream of the 3' half-element of this ERE, and another AP-1-like site was beside the 5' half-element. EMSA analysis using nuclear extracts of E(2)-treated cells and natural sequences, including these putative EREs, indicated that ERbeta - the only isoform expressed in U-937 cells - specifically recognized both EREs because ERbeta-DNA complexes were efficiently competed by the corresponding unlabelled probe and supershifted by the anti-human ERbeta (L-20) antibody. These data provide the first identification of EREs complexed with AP-1-like sites in the hIR promoter, which account for the transcriptional repression of the hIR gene mediated by ERbeta in U-937 cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Complutense University, 28040 Madrid, Spain.
authors.forename: Moisés
authors.initials: M
authors.lastname: García-Arencibia
authors.forename: Norma
authors.initials: N
authors.lastname: Dávila
authors.forename: Javier
authors.initials: J
authors.lastname: Campión
authors.forename: M
authors.initials: M
authors.lastname: Carmen Carranza
authors.forename: Consuelo
authors.initials: C
authors.lastname: Calle
mesh_terms: D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D004958:Estradiol / Q000494:pharmacology; D047629:Estrogen Receptor beta / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics; D011972:Receptor, Insulin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection; D020298:U937 Cells; D014819:Vitellogenins / Q000235:genetics; D014981:Xenopus / Q000235:genetics; D029867:Xenopus Proteins / Q000235:genetics
chemical_list: D047629:Estrogen Receptor beta; D020033:Protein Isoforms; D014819:Vitellogenins; D029867:Xenopus Proteins; D004958:Estradiol; D011972:Receptor, Insulin