IDENTIFICATION OF DEOXYRIBONUCLEIC-ACID SEQUENCES THAT BIND RETINOID-X RECEPTOR-GAMMA WITH HIGH-AFFINITY

The retinoid-X receptor (RXR) family (-alpha, -beta and -gamma) forms homodimers that bind to a number of retinoid-X response elements and t rans-activate gene expression in a retinoid-dependent manner. Although , the RXRs are known to bind tandem direct repeats (DR) of the hexamer , RGGTCA; separated by 1 nucleotide, it is not known whether these rep resent the optimal and/or only recognition sequences. We, therefore, u sed a nonbiased strategy to identify sequences that efficiently bound RXR gamma, an isoform preferentially expressed in cardiac and skeletal muscle tissue. We performed binding site selection with bacterially e xpressed RXR gamma bound to glutathione-agarose and a pool of random s equences to derive a consensus DNA-binding site for RXR gamma. We anal yzed a total of 41 individually selected oligonucleotides and found th at RXR gamma bound with high affinity to motifs that were accommodated by the consensus AARGRNCAAAGGTCA(A)/(C)R. We observed that the majori ty of the sequences that formed complexes with RXR gamma in electropho retic mobility shift analysis were DR-1 motifs; however, DR- motifs se parated by 2, 4, and 8 nucleotides and a palindrome-0 motif were also demonstrated to interact with RXR gamma. Mutagenesis of the derived se quences indicated that both RGGTCA motifs were required for high affin ity binding to RXR gamma. These derived sequences conferred appropriat e 9-cis- and all-trans-retinoic acid (RA) responses to a thymidine kin ase promoter. Furthermore, supershift experiments with a RXR antibody verified that these sequences specifically interacted with RXR in nucl ear extracts derived from C2C12 muscle cells. In conclusion, this stud y rigorously defines the range of DR motifs that can recognize RXR and regulate gene expression in a RA-dependent fashion. The derived conse nsus accommodates retinoid-X response elements that have been identifi ed in a diverse range of genes trans-activated by 9-cis-RA via the RXR family.