Nucleic Acids Research, 2000, Vol. 28, No. 10 2091-2098
© 2000 Oxford University Press
Functional interactions between an atypical NF-
B site from the rat CYP2B1 promoter and the transcriptional repressor RBP-J/CBF1
Department of Molecular and Cell Biology, The University of Texas at Dallas, 2601 North Floyd Road, Richardson, TX 75080, USA
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-
B-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-B and RPB-J/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-B element, but not the Ig NF-B element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-J overexpressed in COS-7 cells repressed expression in vivo from an SV40–luciferase reporter construct that contained the CYP2B1/2 NF-B element. Finally, we observe similar levels of NF-B and RBP-J binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-J/CBF1 binds an atypical NF-B site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.
* To whom correspondence should be addressed. Tel: +1 972 883 6882; Fax: +1 972 231 0628; Email: dejong@utdallas.edu Present address: Xiao-li Wang, Program in Cancer Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
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