In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells.

Abstract

The steroid hormone vitamin D is a principal mediator of skeletal homeostasis. 1,25-Dihydroxyvitamin D3 treatment of ROS 17/2.8 osteoblast-like cells results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. This transcriptional upregulation requires the positive cis-acting vitamin D responsive element (VDRE). We have used the ligation-mediated polymerase chain reaction to demonstrate that protein occupancy of the VDRE within the intact cell correlates with increased synthesis of osteocalcin transcripts. These protein-DNA contacts were not present in the absence of vitamin D or in osteosarcoma cells (ROS 24 Rodan (Merck Sharp & Dohme). Cells were maintained in F12 medium containing calcium and charcoal stripped 5% fetal calf serum for 5 days after plating before hormone treatment or vehicle control. 1,25(OH)2D3 was provided by Milan Uskokovic (Hoffman-La Roche).In Vivo Footprint Analysis. In vivo footprinting was performed by exposing cells to 0.1% dimethy sulfate (DMS) (Aldrich) in culture medium for 5 min at room temperature. Methylated genomic DNA was isolated and cleaved with piperidine (Sigma) (26), and the level of methylation protection of G residues was analyzed by the ligation-mediated single-sided polymerase chain reaction (LMPCR) footprinting technique (27). LMPCR was performed with Vent polymerase (New England Biolabs). First-strand synthesis was accomplished with primer 1 (5'-GACAAACTGGCTCCAAC-TCGCATAGC-3'; nt -358 to -383) in first-strand buffer [40 mM NaCl/10 mM Tris HCl, pH 8.9/4 mM MgSO4/0.01% nonacetylated bovine serum albumin (BSA) (New England Biolabs)/0.1% Triton X-100]. The DNA was ligated with the unidirectional linker primer (27). The DNA was amplified in amplification buffer (10 mM NaCl/20 mM Tris HCl, pH 8.9/2 mM MgSO4/0.01% nonacetylated BSA/0.1% Triton X-100) using primer 2 (5'-GCATAGCCTGTGATTTTCAGTGTCT-GCCGT-3'; nt -377 to -406) and the linker oligonucleotide. Eighteen cycles of PCR were performed, each involving denaturation at 96°C, annealing at 56°C, and elongation at 76°C as described (27). Labeling was performed with 32p-end-labeled primer 3 (5'-AGCCTGTGATTTTCAGTGTCTAbbreviations: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; VDRE, vitamin D responsive element; DMS, dimethyl sulfate; LMPCR, ligation-mediated single-sided PCR.

Inhibition of ligand induced promoter occupancy in vivo by a dominant negative RXR

“…Truss et al (1994), by studying occupancy of the PR remains in vivo, showed that antiprogestins inhibit PR occupancy in the MMTV promoter in vivo, even though antagonist-bound PR was capable of binding to the PRE in vitro. A ligand induced change in VDRE occupancy in vivo has also been noted (Breen et al 1994).…”

The Nuclear Vitamin D Receptor: Biological and Molecular Regulatory Properties Revealed

“…However, unlike the rat osteocalcin gene, where liganded VDR has been shown to associate with the positive VDRE via in vivo footprinting, (160) no evidence has been provided that VDR directly binds to the IL-2 promoter in a ligand-dependent fashion, in vivo.…”

Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation

“…Among the primary VD responsive genes, i.e. genes carrying a functional VDRE, we have studied the expression of the hOC (Breen et al, 1994) and the p21 waf-1 gene (Liu et al, 1996). Among the early responsive gene associated with the monocytic differentiation program we have studied the expression of CD14 (Zhang et al, 1994) and HMSE-1 (Uphoff et al, 1993).…”