Delineation of a DNA recognition element for the vitamin D3 receptor by binding site selection

The vitamin D3 receptor is a ligand-inducible transcriptional regulatory protein. The receptor modulates the transcription of target genes by binding directly to specific DNA sites, termed vitamin D response elements; these sites vary considerably in their homologies to each other. In order to approach the question of what sequences can constitute high affinity recognition elements for the vitamin D3 receptor, we have selected for such sites in vitro by mixing overexpressed and purified vitamin D3 receptor DNA binding domain with an oligonucleotide duplex pool containing a completely randomized central region flanked by primer-annealing sites. Following multiple rounds of immunoprecipitation and amplification by PCR to enrich for high affinity sites, individual clones were sequenced and found to contain nearly identical hexameric sequences, yielding a consensus 5'-AGGGGG-3'. This sequence is similar to some known vitamin D3 receptor binding sites, such as osteocalcin, but quite divergent from others. This suggests that the vitamin D3 receptor may be able to selectively recognize at least two classes of sequence elements.