Figure 5.
Functional analysis of the DRIP complex in a VDR/1,25(OH)2D3-responsive in vitro transcription assay. (A) Depletion experiment: Flow-through fractions run over GST–VDR LBD columns in the absence (Flow − D3; lanes 1–3) or presence (Flow + D3; lanes 4–6) of 1,25(OH)2D3 were used as transcription extracts and tested for their ability to support RXR–VDR activated transcription from a VDRE-linked E1B promoter G-less cassette template. RXR–VDR (25 ng) or 25 ng of RXR–VDR plus 10−7 m 1,25(OH)2D3 was added to each flowthrough. Transcription was allowed to proceed for 45 min, and radiolabeled transcripts 105 nucleotides in length (Test) recovered and separated on a 6% denaturing polyacrylamide gel. A reference E1B promoter-containing G-less cassette of 200 bases devoid of VDREs was included in each reaction as an internal control (Ref). (n.s.) Nonspecific transcript consistently seen in our assay (Lemon et al. 1997). 32P-End-labeled DNA recovery control (Rec.) and molecular mass markers (M) are also shown. (B) Co-eluted GST–VDR–DRIP complex potentiates VDR–RXR transactivation in vitro. Full-length GST–VDR was immobilized on glutathione—Sepharose beads, and nuclear extracts or GST-binding buffer were run over liganded columns as described for the LBD derivative. VDR–DRIP complexes were eluted with 15 mm reduced glutathione and added directly to 25 μg of transcription extracts supplemented with 20 ng of exogenously added RXR plus 10−7 m 1,25(OH)2D3 (lanes 1–8). In vitro transcription assays were carried out as described in A. (Lanes 1,8) No GST-VDR added; (lanes 2–4) 25, 50, and 100 ng of GST–VDR in the absence of DRIPs; (lanes 5–7) 25, 50, and 100 ng GST–VDR in the presence of bound DRIPs. Equivalent amounts of eluted GST–VDR with or without accompanying DRIPs was determined by SDS-PAGE (data not shown). In both A and B, quantitation data is from digitally scanned autoradiographs. Fold transcription (test/reference) were relative to basal transcription (lanes 1,8) set at a value of 1.0.