1. Identification of CAR/RXRα Hetero-dimer Binding Sites in the Human Genome by a Modified Yeast One-Hybrid Assay
- Author
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Jun Inajima, Yuichiro Kanno, Kazuyuki Yanai, Yoshio Inouye, Masashi Sato, and Kenta Hosoda
- Subjects
Genetics ,Nuclear receptor ,Inverted repeat ,Constitutive androstane receptor ,Direct repeat ,Pharmacology (medical) ,Biology ,Binding site ,Retinoid X receptor ,Gene ,Transcription factor - Abstract
The constitutive androstane receptor (CAR) is a transcription factor that belongs to the nuclear receptor superfamily. CAR binds as a heterodimer with the retinoid X receptor α (RXRα) to CAR response elements (CAREs) and regulates the expression of various drug metabolizing enzymes and transporters. To identify CAR/RXRα binding sites in the human genome, we performed a modified yeast one-hybrid assay that enables rapid and efficient identification of genomic targets for DNA-binding proteins. DNA fragments were recovered from positive yeast colonies by PCR and sequenced. A motif enrichment analysis revealed that the most frequent motif was a direct repeat (DR) of RGKTCA-like core sequence spaced by 4 bp. Next, we predicted 149 putative CAR/RXRα binding sites from 414 unique clones, by searching for DRs, everted repeats (ERs) and inverted repeats (IRs) of the RGKTCA-like core motif. Based on gel mobility shift assays, the CAR/RXRα heterodimer could directly interact with the 108 predicted sequences, which included not only classical CAREs but also a wide variety of arrangements. Furthermore, we identified 17 regulatory polymorphisms on the CAR/RXRα-binding sites that may influence individual variation in the expression of CAR-regulated genes. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of CAR/RXRα het-erodimers.
- Published
- 2015