Molecular Endocrinology, Vol 10, 196-205, Copyright © 1996 by Endocrine Society

ARTICLES

Human vitamin D receptor-dependent transactivation in Saccharomyces cerevisiae requires retinoid X receptor

CH Jin and JW Pike
Department of Skeletal Biology, Ligand Pharmaceuticals Inc., San Diego, California 92121, USA.

Transcriptional and DNA binding activities of the human vitamin D receptor (hVDR) were examined in the yeast Saccharomyces cerevisiae. In the studies described here, VDR itself exhibited little transcriptional activity regardless of the nature of the vitamin D-responsive elements (VDREs) used. Consistent with its lack of functional activity, recombinant VDR was unable to bind to VDREs in vitro using bandshift analysis. Interestingly, VDR was able to bind to VDREs with high affinity and to fully activate transcription in intact yeast cells in the presence of the retinoid X receptor (RXR). Although RXR subtypes displayed a similar capacity to induce heterodimer formation with VDR on VDREs, RXR gamma was the strongest of the subtypes in potentiating VDR-dependent transactivation. We also evaluated both DNA binding and transcriptional activities of VDR alone and VDR plus RXR on directly repeated response elements whose half-sites were separated by three and six base pairs. DNA-binding assays together with functional assays revealed that VDR was active only in the presence of RXR, regardless of spacing. Using a domain-swap approach, we constructed a chimeric receptor in which the DNA-binding domain of VDR was replaced with that of the glucocorticoid receptor. Interaction of both wild type and chimeric receptors with a hybrid-responsive element in the presence of RXR revealed that RXR and VDR bound to the 5'- and 3'-half-sites of VDRE, respectively. Finally, we show that the fifth position in the 3'- half-site (C) of the VDRE strongly influences the binding of VDR/RXR heterodimer to its cognate cis-elements. Cumulatively, our studies demonstrate, using an eukaryotic yeast system, that the functional VDR unit includes RXR or an equal partner.

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