Identification of CAR/RXR<i>α</i>Hetero-dimer Binding Sites in the Human Genome by a Modified Yeast One-Hybrid Assay

作     者：Kenta Hosoda Yuichiro Kanno Masashi Sato Jun Inajima Yoshio Inouye Kazuyuki Yanai 

作者机构：Department of Biomolecular Science Faculty of Science Toho University Chiba Japan Faculty of Pharmaceutical Sciences Toho University Chiba Japan 

出 版 物：《Advances in Biological Chemistry》 (生物化学进展（英文）)

年 卷 期：2015年第5卷第2期

页      面：83-97页

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

主　　题：Constitutive Androstane Receptor Retinoid X Receptor Transcription SNP Polymorphism Nuclear Receptor 

摘      要：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.