Rat CYP1A1 negative regulatory element: biological activity and interaction with a protein from liver and hepatoma cells.
Rat CYP1A1 promoter activity was suppressed by the presence of a cis negative regulatory element (NRE) at position -843 to -746 in transiently transfected rat H4IIE and human HepG2 hepatoma cells. Removal of the NRE from the promoter-fusion gene constructs caused an increase in the basal promoter activity of 2-6-fold. Co-transfection of the NRE-containing or non-NRE-containing CYP1A1 promoter-fusion gene constructs with a cloned rat NRE, i.e., pNRE, into HepG2 cells caused a 2-fold or greater reduction in constitutive and induced promoter activities. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced expression of the endogenous human CYPA1 was also inhibited by transfection of pNRE into HepG2 cells. Deletion of the sequence from base pairs (bp) -658 to -269 in the NRE-containing construct caused a dramatic decrease of constitutive expression in transiently transfected HepG2 cells, compared with an identical construct that lacked the NRE. Deletion of the sequences between bp -658 and -158 in the CYP1A1 promoter did not affect reporter gene activity, indicating a second site of interaction. At least three different rat liver nuclear proteins bound to the rat NRE, as determined by gel mobility shift and DNase I footprinting assays. A 32-bp sequence within the rat NRE, with significant sequence identity to the 26-bp c-myc, fos/jun-octamer-binding, NRE, was protected from DNAse I cleavage by rat liver nuclear extracts. These data suggested a role for this region in the negative regulation of rat CYP1A1.
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.
K
K
Sterling
J
J
Weaver
K L
KL
Ho
L C
LC
Xu
E
E
Bresnick
D000818:Animals; D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D011993:Recombinant Fusion Proteins; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System