|Completion of the human genome project led to the post-genome era. Next
important task is to apply differences of genome information of each individual
to the personalized medicine. Non-synonymous single nucleotide polymorphisms
(SNPs) that result in amino acid changes in proteins were extensively studied.
However examples of variation in regulatory sequences are poorly reported,
although there are several examples of regulatory SNPs associated with disease susceptibility related to development and differentiation.
Here, we focused on the constitutive androstane receptor (CAR) which is
a transcription factor that belongs to the nuclear receptor superfamily.
CAR binds as a heterodimer with the retinoid X receptor ƒ¿ (RXRƒ¿) to CAR
response elements (CAREs) and regulates the expression of various drug
metabolizing enzymes and transporters.To identify CAR/RXRƒ¿ binding sites
in the human genome, we performed a modified yeast one-hybrid system [J Recept Signal Transduct Res. 2010 Apr;30(2):88-105, Methods Mol Biol. 2013;977:125-36] . The human genomic 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/RXRƒ¿ 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/RXRƒ¿ 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/RXRƒ¿-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/RXRƒ¿ heterodimers.