Vitamin D receptor interactions with positive and negative DNA response elements: an interference footprint comparison

Interference footprinting protocols were utilized to examine the interactions of the vitamin D receptor (VDR) with either a positive or a negative vitamin D response element (VDRE). A sequence from the human osteocalcin (hOC) gene was chosen for the prototypical positive DR+3 VDRE, while an analogous sequence linked to the avian parathyroid hormone gene (aPTH) was used as the negative VDRE. Both types of response elements were examined for phosphate backbone contacts, as well as base-specific interactions with guanine and thymine residues. Sources of VDR included partially purified canine intestinal preparations, as well as extracts of recombinant human VDR and retinoid X receptor alpha prepared from baculovirus-infected Sf9 insect cells. Cold competition experiments using variable amounts of these oligonucleotides in the mobility shift assay revealed that the hOC element was a five-fold better competitor for heterodimer complex binding than the negative VDRE. Interference footprints revealed extensive strong contacts to the phosphate backbone and individual guanine and thymine nucleotides of the hOC element. The composite hOC footprint was asymmetric for the number and strength of interactions observed over each of the respective direct repeat half-sites. In contrast, the aPTH VDRE footprints revealed fewer points of DNA contact that were limited to the hexanucleotide repeat regions and were strikingly weaker in nature. The alignment of DNA contact points for both elements produced a 5' stagger that was indicative of successive major groove interactions, and consistent with dimer binding. DNA helical representations indicate that the heterodimer contacts to these response elements are substantially different and provide insight into functional aspects of each complex.     

The vitamin D response element-binding protein. A novel dominant-negative regulator of vitamin D-directed transactivation

Vitamin D resistance in certain primate genera is associated with the constitutive overexpression of a non-vitamin D receptor (VDR)-related, vitamin D response element-binding protein (VDRE-BP) and squelching of vitamin d-directed transactivation. We used DNA affinity chromatography to purify proteins associated with non-VDR-VDRE binding activity from vitamin d-resistant New World primate cells. In electrophoretic mobility shift assays, these proteins bound specifically to either single-strand or double-strand oligonucleotides harboring the VDRE. Amino acid sequencing of tryptic peptides from a 34-kDa (VDRE-BP1) and 38-kDa species (VDRE-BP-2) possessed sequence homology with human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2, respectively. cDNAs bearing the open reading frame for both VDRE-BPs were cloned and used to transfect wild-type, hormone-responsive primate cells. Transient and stable overexpression of the VDRE-BP2 cDNA, but not the VDRE-BP1 cDNA, in wild-type cells with a VDRE-luciferase reporter resulted in significant reduction in reporter activity. These data suggest that the hnRNPA2-related VDRE-BP2 is a dominant-negative regulator of vitamin D action.     

Vitamin D receptor interactions with the murine osteopontin response element

The nature of the DNA binding interactions of the human vitamin D receptor (hVDR) with the murine osteopontin vitamin D response element (mOP VDRE) was examined. Both recombinant hVDR and human retinoid X receptor β (hRXRβ) proteins were obtained from baculovirus-infected Sf9 insect cells. Mixing extracts of the two recombinant proteins resulted in the strong, specific formation of a slower migrating complex in the electrophoretic mobility shift assay. Crude extracts of the expressed hVDR alone were also capable of binding with high affinity to the mOP sequence, and this binding was enhanced in the presence of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃). Competition experiments confirmed the specificity of this interaction and revealed that the human osteocalcin VDRE was a poor competitor for this binding. Ethylation interference footprint analyses of hVDR/hRXRβ and hVDR complexes revealed only subtle differences in how these two different VDR-containing complexes interacted with the mOP VDRE. The footprints displayed contact points in both halves of the direct repeat format, confirming the dimeric and major groove interactions of both types of complexes. DNA affinity chromatography of labelled hVDR extracts revealed a peak eluting at ca. 290 mM KCl that was capable of rebinding to the mOP sequence in gel shift experiments. Ultraviolet (UV) light-crosslinking experiments of hVDR extracts alone to radiolabelled DNA were consistent with the existence of a homodimeric hVDR interaction. Additionally, these experiments confirmed the direct interaction of a hVDR/hRXRβ heterodimer when mixed extracts were utilized. From these results we infer that homodimers of the hVDR which respond with enhanced DNA binding to particular vitamin D response elements when exposed to 1,25-(OH)₂D₃ are possible. This may be of functional significance when RXR proteins are limiting or RXR ligand is present within a cell.     

Investigation on the Potential Role of the AH Receptor Nuclear Translocator Protein in Vitamin D Receptor Action

Abstract

The Ah receptor nuclear translocator protein (ARNT) is required for binding of the Ah (dioxin) receptor to the xenobiotic responsive element (XRE), and is a structural component of the XRE-binding form of the Ah receptor. The vitamin D receptor requires an accessory protein for binding to the vitamin D responsive element (VDRE) in the osteocalcin gene. Since the vitamin D receptor has similarities to the Ah receptor, we investigated whether ARNT is also required for vitamin D receptor activity. Two lines of evidence demonstrate that ARNT is not required for vitamin D receptor activity, and therefore does not correspond to the vitamin D receptor accessory protein: i) Antibodies to ARNT have no effect on binding of the vitamin D receptor to the VDRE. ii) c4, a mutant of Hepa-1 cells that is defective in ARNT activity, and in which binding of the Ah receptor to the XRE does not occur, possesses a vitamin D receptor with full activity for binding the VDRE.     

A 55-kilodalton accessory factor facilitates vitamin D receptor DNA binding.

The interaction of the vitamin D receptor with a vitamin D-responsive element (VDRE) derived from the human osteocalcin promoter in vitro has been shown to require a nuclear accessory factor (NAF) derived from monkey kidney cells. In this report we show that this factor is widely distributed in cells and tissues, including those that do not express the vitamin D receptor (VDR). NAF is required for VDR binding to a variety of known VDREs. VDR and NAF independently bind the VDRE weakly, as assessed by elution profiles generated during VDRE affinity chromatography. Together, however, both proteins coelute from this column with a profile that indicates a tighter strength of interaction. Analogous chromatography of the VDR derived from ROS 17/2.8 cells treated with 1,25-dihydroxyvitamin D3 in culture also reveals a dual profile of weak and strong binding, suggesting that in vivo modifications are unlikely to alter receptor DNA binding. NAF is a protein of 55 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cross-linking experiments suggest that the VDR and NAF together form a heterodimer on a single VDRE with a mol wt of 103 kDa. These data demonstrate that NAF is required for VDR binding to specific DNA in vitro and suggest the possibility that NAF may be required for the transactivation capability of the VDR in vivo.     

Cross-talk between 1,25-dihydroxyvitamin D3 and transforming growth factor-beta signaling requires binding of VDR and Smad3 proteins to their cognate DNA recognition elements

1,25-Dihydroxyvitamin D(3) (vitamin D) and transforming growth factor-beta (TGF-beta) regulate diverse biological processes including cell proliferation and differentiation through modulation of the expression of target genes. Members of the Smad family of proteins function as effectors of TGF-beta signaling pathways whereas the vitamin D receptor (VDR) confers vitamin D signaling. We investigated the molecular mechanisms by which TGF-beta and vitamin D signaling pathways interact in the regulation of the human osteocalcin promoter. Synergistic activation of the osteocalcin gene promoter by TGF-beta and vitamin D was observed in transient transfection experiments. However, in contrast to a previous report by Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K., and Kato, S. (1999) Science, 283, 1317-1321, synergistic activation was not detectable when the osteocalcin vitamin D response element (VDRE) alone was linked to a heterologous promoter. Inclusion of the Smad binding elements (SBEs) with the VDRE in the heterologous promoter restored synergistic activation. Furthermore, this synergy was dependent on the spacing between VDRE and SBEs. The Smad3-Smad4 heterodimer was found to bind in gel shift assay to two distinct DNA segments of the osteocalcin promoter: -1030 to -989 (SBE3) and -418 to -349 (SBE1). Deletion of SBE1, which is proximal to the VDRE, but not the distal SBE3 in this promoter reporter abolished TGF-beta responsiveness and eliminated synergistic co-activation with vitamin D. Thus the molecular mechanism, whereby Smad3 and VDR mediate cross-talk between the TGF-beta and vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter.     

Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] plays a critical role in maintaining calcium and phosphate homeostasis and bone formation but also exhibits antiproliferative activity on many cancer cells, including prostate cancer. We have shown that the antiproliferative actions of 1,25-(OH)2D3 in the LNCaP human prostate cancer cell line are mediated in part by induction of IGF binding protein-3 (IGFBP-3). The purpose of this study was to determine the molecular mechanism involved in 1,25-(OH)2D3 regulation of IGFBP-3 expression and to identify the putative vitamin D response element (VDRE) in the IGFBP-3 promoter. We cloned approximately 6 kb of the IGFBP-3 promoter sequence and demonstrated its responsiveness to 1,25-(OH)2D3 in transactivation assays. Computer analysis identified a putative VDRE between -3296/-3282 containing the direct repeat motif GGTTCA ccg GGTGCA that is 92% identical with the rat 24-hydroxylase distal VDRE. In EMSAs, the vitamin D receptor (VDR) showed strong binding to the putative IGFBP-3 VDRE in the presence of 1,25-(OH)2D3. Supershift assays confirmed the presence of VDR in the IGFBP-3 VDRE complex. Chromatin immunoprecipitation assay demonstrated that 1,25-(OH)2D3 recruited the VDR/retinoid X receptor heterodimer to the VDRE site in the natural IGFBP-3 promoter in intact cells. In transactivation assays, the putative VDRE coupled to a heterologous simian virus 40 promoter construct was induced 2-fold by 1,25-(OH)2D3. Mutations in the VDRE resulted in a loss of inducibility confirming the critical hexameric sequence. In conclusion, we have identified a functional VDRE in the distal region of the human IGFBP-3 promoter. The induction of IGFBP-3 by 1,25-(OH)2D3 appears to be directly mediated via VDR interaction with this VDRE.     

Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression

The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.