Identification of DNA sequences that bind retinoid X receptor-1,25(OH)2D3-receptor heterodimers with high affinity.
Vitamin D3 receptors (VDR) bind as heterodimers with retinoid X receptors (RXR) to vitamin D response elements (VDRE) and transactivate gene expression in a 1,25(OH)2D3-dependent manner. These elements are tandem direct repeats (DRs) of the hexamer RGGTCA separated by three nucleotides (DR3). We determined whether this DR3 was the optimal and/or only recognition sequence, by PCR-mediated binding site selection with reticulocyte lysate-expressed hVDR and mRXRalpha, and a pool of random sequences. We derived a consensus binding site for RXR-VDR heterodimers, RGGTCANN RRGTTCAB, and analyzed 10 of the 45 sequences slected by EMSA, methylation interference and transfection experiments: all the sequences were specific and acted as positive VDREs; the underlined purine of the spacer interacted with the heterodimer; the mutation of the third T in the second motif to a G did not influence VDRE activity. Thus, the selectivity of vitamin D pathway involving heterodimerization rather than VDR-homodimerization is not due to internal sequence variations. Except for mouse osteopontin VDRE, the natural VDREs would be efficient, only when helped by adjacent sequences and/or transactivators other than VDR and RXR.
Colnot
S
S
INSERM U120, Hôpital Robert Debré, Paris, France.
Lambert
M
M
Blin
C
C
Thomasset
M
M
Perret
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002117:Calcitriol / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D018983:DNA Footprinting; D046911:Macromolecular Substances; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D046911:Macromolecular Substances; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D004247:DNA; D002117:Calcitriol