Functional interactions between an atypical NF-kappaB site from the rat CYP2B1 promoter and the transcriptional repressor RBP-Jkappa/CBF1.
The phenobarbital-inducible rat cytochrome P450 (CYP) 2B1 and 2B2 proteins are encoded by homologous genes whose promoters contain a mammalian-apparent long terminal repeat retrotransposon (MaLR). An NF-kappaB-like site within the MaLR forms multiple protein-DNA complexes with rat liver and HeLa cell nuclear extracts. Using antibody supershift assays, we have identified these complexes as NF-kappaB and RPB-Jkappa/CBF1. Competition assays using a series of single site mutant oligonucleotides reveal that the recognition sites for these two factors overlap. We also show that the CYP2B1/2 NF-kappaB element, but not the Igkappa NF-kappaB element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-Jkappa over-expressed in COS-7 cells repressed expression in vivo from an SV40-luciferase reporter construct that contained the CYP2B1/2 NF-kappaB element. Finally, we observe similar levels of NF-kappaB and RBP-Jkappa binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-Jkappa/CBF1 binds an atypical NF-kappaB site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.
Lee
S H
SH
Department of Molecular and Cell Biology, The University of Texas at Dallas, 2601 North Floyd Road, Richardson, TX 75080, USA.
Wang
X
X
DeJong
J
J
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008099:Liver / Q000201:enzymology; D008156:Luciferases / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism*; D018626:Retroelements; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013250:Steroid Hydroxylases / Q000235:genetics; D013698:Templates, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; C497570:RBPJ protein, human; C497572:Rbpjl2 protein, rat; D011994:Recombinant Proteins; D012097:Repressor Proteins; D018626:Retroelements; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C025619:steroid 16-beta-hydroxylase