DNA sequences that act as high affinity targets for the vitamin D3 receptor in the absence of the retinoid X receptor.
DNA binding site discrimination within a subgroup of nuclear receptors, including the human vitamin D3 receptor (hVDR), appears to be influenced primarily by spacing and orientation differences of response element half-sites, since many receptors recognize and bind to the same hexameric half-site sequence, AGGTCA. Small sequence differences within half-sites, however, may also play an important role in distinguishing between different receptor complexes. Several laboratories have reported that the AGGTCA element in a direct repeat (DR) configuration appears 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; essentially the mouse osteopontin [Spp-1] vitamin D response element) acts as a high affinity target for purified hVDR in the absence of RXR. This suggests that some half-site sequences could be targets for hVDR alone while others serve as recognition elements for hVDR-RXR complexes. In this report, we test this hypothesis by selecting, using purified hVDR only, for high affinity receptor binding sites in a complex DNA mixture which should by chance contain such sequences. We find that the purified receptor selects a heptameric sequence resembling a half-site of the osteopontin vitamin D response element, consistent with osteopontin-like sequences acting as high affinity targets for hVDR in the absence of RXR. We directly test this by comparing the in vitro DNA binding activity of purified hVDR to DR+3 elements comprised of osteopontin-like AGTTCA or AGGTCA half-sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Biology & Genetics Program Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
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Freedman
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Arce
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Perez Fernandez
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors
D000345:Affinity Labels; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D018167:Receptors, Calcitriol; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D004247:DNA