ABC  Vol.5 No.2 , April 2015
Identification of CAR/RXRα Hetero-dimer Binding Sites in the Human Genome by a Modified Yeast One-Hybrid Assay
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.
Cite this paper: Hosoda, K. , Kanno, Y. , Sato, M. , Inajima, J. , Inouye, Y. and Yanai, K. (2015) Identification of CAR/RXRα Hetero-dimer Binding Sites in the Human Genome by a Modified Yeast One-Hybrid Assay. Advances in Biological Chemistry, 5, 83-97. doi: 10.4236/abc.2015.52008.

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