DNA-SEQUENCES THAT ACT AS HIGH-AFFINITY TARGETS FOR THE VITAMIN-D-3 RECEPTOR IN THE ABSENCE OF THE RETINOID-X RECEPTOR

DNA binding site discrimination within a subgroup of nuclear receptors , including the human vitamin D-3 receptor (hVDR), appears to be influ enced primarily by spacing and orientation differences of response ele ment half-sites, since many receptors recognize and bind to the same h exameric half-site sequence, AGGTCA. Small sequence differences within half-sites, however, may also play an important role in distinguishin g between different receptor complexes. Several laboratories have repo rted that the AGGTCA element in a direct repeat (DR) configuration app ears to be a high affinity recognition site for only nuclear receptor- 9 retinoid X receptor (RXR) heterodimers. However, we have previously shown that a closely related, but distinct, element (AGTTCA; essential ly the mouse osteopontin [Spp-1] vitamin D response element) acts as a high affinity target for purified hVDR in the absence of RXR. This su ggests that some half-site sequences could be targets for hVDR alone w hile others serve as recognition elements for hVDR-RXR complexes. In t his report, we test this hypothesis by selecting, using purified hVDR only, for high affinity receptor binding sites in a complex DNA mixtur e which should by chance contain such sequences. We find that the puri fied receptor selects a heptameric sequence resembling a half-site of the osteopontin vitamin D response element, consistent with osteoponti n-like sequences acting as high affinity targets for hVDR in the absen ce of RXR. We directly test this by comparing the in vitro DNA binding activity of purified hVDR to DR+3 elements comprised of osteopontin-l ike AGTTCA or AGGTCA half-sites. While an hVDR-RXR heterodimer binds w ith high affinity to both elements, hVDR alone binds only to the AGTTC A DR+3. We show that a T/A base pair at the third position of each hal f-site is of primary importance for homodimeric hVDR recognition. Thes e observations suggest that small sequence differences within half-sit es could be determinants of target site selectivity for homodimeric vs . heterodimer hVDR complexes.