RETINOID X RECEPTOR-VITAMIN-D-3 RECEPTOR HETERODIMERS PROMOTE STABLE PREINITIATION COMPLEX-FORMATION AND DIRECT 1,25-DIHYDROXYVITAMIN-D-3-DEPENDENT CELL-FREE TRANSCRIPTION

The numerous members of the steroid/nuclear hormone receptor superfami ly act as direct transducers of circulating signals, such as steroids, thyroid hormone, and vitamin or lipid metabolites, and modulate the t ranscription of specific target genes, primarily as dimeric complexes. The receptors for 9-cis retinoic acid and 1,25-dihydroxyvitamin D-3 [ 1,25(OH)(2)D-3], RXR and VDR, respectively, as members of this superfa mily, form a heterodimeric complex and bind cooperatively to vitamin D responsive elements (VDREs) to activate or repress the transcription of a multitude of genes which regulate a variety of physiological func tions. To directly investigate RXR- and VDR-mediated transactivation, we developed a cell-free transcription system for 1,25(OH),D, signalin g by utilizing crude nuclear extracts and a G-free cassette-based assa y. Transcriptional enhancement in vitro was dependent on purified, exo genous RXR and VDR and was responsive to physiological concentrations of 1,25(OH)(2)D-3. We found that RXR and VDR transactivated selectivel y from VDRE-linked templates exclusively as a heterodimeric complex, s ince neither receptor alone enhanced transcription in vitro, By the ad dition of low concentrations of the anionic detergent Sarkosyl to limi t cell-free transcription to a single round and the use of agarose gel mobility shift experiments to assay factor complex assembly, we obser ved that 1,25(OH),D, enhanced RXR:VDR-mediated stabilization or assemb ly of preinitiation complexes to effect transcriptional enhancement fr om VDRE-linked promoter-containing DNA.