RETINOID X-RECEPTOR-ALPHA BINDS WITH THE HIGHEST AFFINITY TO AN IMPERFECT DIRECT REPEAT RESPONSE ELEMENT

The regulation of gene expression by retinoids is mediated by two classes of receptors, retinoic acid receptors and retinoid X receptors (RXR). RXR can bind to specific target genes as homodimers, and these homodimers can activate gene expression in the presence of the Ligand 9-cis-retinoic acid. A direct repeat of AGGTCA with a 1 base pair spacer (DR1) acts as a RXR homodimer response element in the presence of 9-cis-retinoic acid. However, it is not known if this represents the highest affinity binding site for the RXR homodimer. To investigate this question, we used a nonbiased strategy to isolate from a pool of random DNA those sequences that have the highest affinity for RXR alpha homodimers. The imperfect DR1 sequence 5'-GGGGTCAAAGGTCA displayed the highest in vitro binding affinity for RXR alpha homodimers. Transient transfection studies confirmed that this sequence is a more potent response element than is a perfect DR1 of either AGGTCA or GGGGTCA. The results also indicate that for RXR alpha homodimers, the receptor hound to the 5' half-site dislays different DNA binding specificity than that bound to the 3' half-site. Thus, DNA binding specificity is determined not only by the amino acid sequence of the protein but also by its protein-protein interactions and its position on the response element (5' vs. 3').