The orphan nuclear receptor DAX-1 functions as a potent corepressor of the constitutive androstane receptor (NR1I3).
Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately with coregulator proteins. The human constitutive androstane receptor (CAR; NR1I3) is expressed primarily in liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy, and lipid homeostasis. In this report, DAX-1, a nuclear receptor family member with corepressor properties, was identified as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1's downstream LXXLL and its PCFQVLP motifs were critical contributors to DAX-1's corepression activities, although two other LXXM/LL motifs located nearer the N terminus had no impact on the CAR functional interaction. Deletion of DAX-1's C-terminal transcription silencing domain restored CAR1 transactivation activity in reporter assays to approximately 90% of control, demonstrating its critical function in mediating the CAR repression activities. Furthermore, results obtained from mammalian two-hybrid experiments assessing various domain configurations of the respective receptors showed that full-length DAX-1 inhibited the CAR-SRC1 interaction by approximately 50%, whereas the same interaction was restored to 90% of control when the DAX-1 transcription silencing domain was deleted. Direct interaction between CAR and DAX-1 was demonstrated with both alpha-screen and coimmunoprecipitation experiments, and this interaction was enhanced in the presence of the CAR activator 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Results obtained in primary human hepatocytes further demonstrated DAX-1 inhibition of CAR-mediated CITCO induction of the CYP2B6 target gene. The results of this investigation identify DAX-1 as a novel and potent CAR corepressor and suggest that DAX-1 functions as a coordinate hepatic regulator of CAR's biological function.
Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Elizabeth M
EM
Laurenzana
Tao
T
Chen
Malavika
M
Kannuswamy
Brian E
BE
Sell
Stephen C
SC
Strom
Yong
Y
Li
Curtis J
CJ
Omiecinski
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics; D056970:Co-Repressor Proteins / Q000378:metabolism*; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D057137:DAX-1 Orphan Nuclear Receptor / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000235:genetics; D010091:Oximes / Q000494:pharmacology; D061251:Primary Cell Culture; D020033:Protein Isoforms / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D056970:Co-Repressor Proteins; D000090702:Constitutive Androstane Receptor; D057137:DAX-1 Orphan Nuclear Receptor; C000716327:NR1I3 protein, human; D010091:Oximes; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D013844:Thiazoles; C475093:6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; D008156:Luciferases; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating