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Oct-1 transcription factor is a negative regulator of rat CYP1A1 expression via an octamer sequence in its negative
1Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, Worcester 01655, USA.
Abstract:
The rat CYP1A1 negative regulatory element (NRE) contains AP-1 and Oct-1 motifs at -808 to -788 bp. The CYP1A1 sequence from -813 to -779 bp and an identical sequence bearing a point mutation in the octamer motif were synthesized. Gel mobility shift assays showed the formation of two complexes with the wild-type CYP1A1 sequence and nuclear extracts from H4IIE and HepG2 hepatoma cells and from rat liver. The formation of the major complex was significantly reduced with the mutant octamer-containing oligomer and was specifically competed by an Oct-1 oligodeoxyribonucleotide. The addition of Oct-1 antibody caused a supershift of the major complex. The presence of the wild-type sequence, but not the mutant octamer sequence, caused a 3-fold decrease in SV40 enhancerless promoter activity in transfected HepG2 cells. Co-transfection of an Oct-1 expression vector with rat CYP1A1 NRE octamer-containing, promoter/reporter gene constructs specifically further decreased promoter activity of the wild-type octamer-containing constructs in HepG2 cells. The results indicate that Oct-1 binds to the rat CYP1A1 promoter NRE and is a negative regulator of rat CYP1A1 expression.
Insights
Oct-1 binds to the rat CYP1A1 promoter's negative regulatory element, acting as a repressor. This binding significantly reduces CYP1A1 gene expression in liver cells.
Area of Science:
- Molecular Biology
- Gene Regulation
- Hepatology
Background:
- The rat cytochrome P450 1A1 (CYP1A1) gene expression is tightly regulated.
- A negative regulatory element (NRE) in the CYP1A1 promoter contains binding sites for transcription factors.
Purpose of the Study:
- To investigate the role of the Oct-1 transcription factor in regulating rat CYP1A1 gene expression.
- To characterize the interaction of Oct-1 with the CYP1A1 NRE.
Main Methods:
- Gel mobility shift assays using synthesized wild-type and mutant CYP1A1 NRE sequences with nuclear extracts.
- Reporter gene assays in transfected HepG2 cells to assess promoter activity.
- Supershift assays with Oct-1 antibody and competition assays with Oct-1 oligodeoxyribonucleotides.
Main Results:
- Oct-1 specifically binds to the wild-type CYP1A1 NRE, forming a major complex in nuclear extracts.
- Mutation of the Oct-1 motif significantly reduced complex formation.
- The wild-type NRE, but not the mutant, decreased promoter activity, an effect further enhanced by Oct-1 co-transfection.
Conclusions:
- Oct-1 binds to the rat CYP1A1 promoter NRE.
- Oct-1 functions as a negative regulator of rat CYP1A1 gene expression.