Characterization of DNA complexes formed by the nuclear receptor constitutive androstane receptor.
The nuclear receptor constitutive androstane receptor (CAR) acts as a xenobiotic sensor and regulates the expression of enzymes, such as several cytochromes P450s and the UDP-glucuronosyltransferase (UGT) type 1A1. CAR binds as a heterodimer with the retinoid X receptor (RXR) to specific DNA sites, called response elements (REs). Clusters of CAR REs, referred to as phenobarbital response enhancer modules (PBREMs), have been identified in several CAR target genes. In this study we confirm that REs formed by direct repeats of two AGTTCA hexamers with 4 spacing nucleotides are optimal for the binding of CAR-RXR heterodimers. In addition, we found that the heterodimers also form complexes on everted repeat-type arrangements with 8 spacing nucleotides. We also observed that CAR is able to bind DNA as a monomer and to interact in this form with different coregulators even in the presence of RXR. Systematic variation of the nucleotides 5'-flanking to both AGTTCA hexamers showed that the dinucleotide sequence modulates the DNA complex formation of CAR monomers and CAR-RXR heterodimer by a factor of up to 20. The highest preference was found for the sequence AG and lowest for CC. The increased DNA affinity of CAR is mediated by the positively charged arginines 90 and 91 located in the carboxyl-terminal extension of the DNA-binding domain of the receptor. Furthermore, we show that one of the three CAR REs of the human UGT1A1 PBREM is exclusively bound by CAR monomers and this is regulated by ligands that bind to this nuclear receptor. This points to a physiological role for CAR monomers. Therefore, both CAR-RXR heterodimers and CAR monomers can contribute to the gene activating function of PBREMs in CAR target genes.
Frank
Christian
C
Department of Biochemistry, University of Kuopio, FIN-70211 Kuopio, Finland.
Gonzalez
Manuel Macias
MM
Oinonen
Carita
C
Dunlop
Thomas W
TW
Carlberg
Carsten
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D002645:Chickens; D000090702:Constitutive Androstane Receptor; D004247:DNA / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D000090702:Constitutive Androstane Receptor; D018076:DNA, Complementary; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase