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Rat CYP1A1 negative regulatory element: biological activity and interaction with a protein from liver and hepatoma
1Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.
Abstract:
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.
Insights
A negative regulatory element (NRE) in the rat CYP1A1 promoter suppresses gene activity. This NRE binds nuclear proteins, suggesting its role in regulating CYP1A1 expression, particularly in response to dioxin induction.
Area of Science:
- Molecular Biology
- Gene Regulation
- Biochemistry
Background:
- The Cytochrome P450 1A1 (CYP1A1) gene is crucial for metabolizing xenobiotics.
- Understanding the regulation of CYP1A1 is vital for predicting and mitigating toxicological effects.
- Previous studies indicated complex regulatory mechanisms for CYP1A1.
Purpose of the Study:
- To identify and characterize a negative regulatory element (NRE) in the rat CYP1A1 promoter.
- To investigate the role of the NRE in both basal and induced CYP1A1 expression.
- To elucidate the molecular interactions involved in NRE-mediated repression.
Main Methods:
- Transient transfection assays using promoter-luciferase constructs in H4IIE and HepG2 cells.
- Deletion analysis of the CYP1A1 promoter region.
- Gel mobility shift assays and DNase I footprinting to identify protein-DNA interactions.
- Co-transfection experiments with cloned NRE sequences.
Main Results:
- A cis-acting NRE located at -843 to -746 bp significantly suppressed rat CYP1A1 promoter activity.
- Removal of the NRE increased basal promoter activity 2-6 fold.
- Introduction of a cloned NRE reduced both constitutive and induced promoter activities by at least 2-fold.
- The NRE also inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced expression of endogenous human CYP1A1.
- Deletion analysis identified a critical region within the NRE (-658 to -269 bp) and suggested a second interaction site.
- At least three nuclear proteins bound to the NRE, with a 32-bp sequence showing homology to known octamer-binding sites.
Conclusions:
- A novel NRE in the rat CYP1A1 promoter plays a significant role in negative gene regulation.
- This NRE functions by binding multiple nuclear proteins, including those interacting with a conserved octamer-binding motif.
- The NRE contributes to the repression of both basal and induced CYP1A1 expression, impacting xenobiotic metabolism and toxicity.