TRANSCRIPTIONAL SYNERGISM BETWEEN THE VITAMIN-D-3 RECEPTOR AND OTHER NONRECEPTOR TRANSCRIPTION FACTORS

Small changes in the concentrations and/or combinations of trans-actin g factors can result in profound alterations in gene expression. Syner gistic interaction between different classes of transcription factors bound to distinct sites within a promoter/enhancer region is one mecha nism by which this can occur. Reflecting this, hormone response elemen ts, DNA recognition sites for steroid/nuclear receptors, are often fou nd in promoter regions organized as multiple copies or are clustered a mong binding sites for other trans-acting factors. To systematically e xamine the potential interactions between one such receptor, the vitam in D-3 receptor (VDR), and other nonreceptor transcription factors, we constructed a series of reporter plasmids containing one copy of the osteopontin (Spp1) vitamin D response element (VDRE), consisting of tw o direct repeats spaced by 3 base pairs, and one binding site for the transcription factors SP1, NF-1, Oct-1, or AP-1. We also generated rep orters either under the control of two copies of Spp1 VDRE, or a disti nct VDRE from the human osteocalcin gene promoter. The various reporte rs were used to transiently transfect HeLa or CV-1 cells in the presen ce and absence of 1,25-dihydroxyvitamin D-3. Our results show that VDR transactivates 12-20 times more strongly from two Spp1-VDREs than fro m one, indicating that VDR synergizes with itself. VDR also synergizes with the other nonreceptor factors, since we observe a 6- to le-fold degree of synergistic induction after ligand addition, depending on th e particular factor. The functional basis for the transcriptional syne rgism appears to be at the level of cooperative DNA binding, at least for VDR alone and VDR-Oct-1, as demonstrated in vitro by gel mobility shift assays using purified factors. Consistent with this, we show tha t the minimal requirement for transcriptional synergism in vivo by VDR is its DNA-binding domain.