Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter.
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.
García-Arencibia
Moisés
M
Department of Biochemistry and Molecular Biology, School of Medicine, Complutense University, 28040 Madrid, Spain.
Dávila
Norma
N
Campión
Javier
J
Carmen Carranza
M
M
Calle
Consuelo
C
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
D047629:Estrogen Receptor beta; D020033:Protein Isoforms; D014819:Vitellogenins; D029867:Xenopus Proteins; D004958:Estradiol; D011972:Receptor, Insulin