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1.
J Cell Biochem ; 69(1): 1-12, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9513041

ABSTRACT

The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes. Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D. Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR. Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937. Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast. We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element. Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid. Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR. Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements.


Subject(s)
Receptors, Calcitriol/metabolism , Receptors, Retinoic Acid/metabolism , Ribosomal Proteins/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Base Sequence , Binding Sites/genetics , Cell Line , DNA Primers/genetics , Dimerization , Enhancer Elements, Genetic , Escherichia coli/genetics , Escherichia coli Proteins , Humans , In Vitro Techniques , Ligands , Protein Binding , Receptors, Calcitriol/chemistry , Receptors, Calcitriol/genetics , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retinoid X Receptors , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Saccharomyces cerevisiae/genetics , Sequence Deletion , Transcription Factors/chemistry , Transcription Factors/genetics , Vitamin D-Binding Protein/chemistry , Vitamin D-Binding Protein/genetics , Vitamin D-Binding Protein/metabolism
2.
J Cell Biochem ; 66(2): 184-96, 1997 Aug 01.
Article in English | MEDLINE | ID: mdl-9213220

ABSTRACT

The transcription factors of the nuclear hormone receptor family regulate gene expression via a complex network of macromolecular interactions. The ligand dependent activity of the vitamin D receptor is of particular interest because it modulates gene expression by the heterodimeric interaction with retinoid X receptors. We report here that individual functions of the vitamin D receptor including DNA-binding, homo- and heterodimerization and transactivation can be reconstituted in the yeast Saccharomyces cerevisiae. Interestingly, the simultaneous expression of the native vitamin D receptor and the retinoid X receptor beta resulted in a ligand independent transactivation of the lacZ reporter gene coupled to a mouse osteopontin vitamin D response element. However, homodimerization of the vitamin D receptor and heterodimerization were strongly enhanced upon ligand binding, when the receptors were expressed as fusion proteins with the Gal4 transcription factor in a yeast two-hybrid system. Furthermore, transactivating activity of a Gal4-fused vitamin D receptor was induced by vitamin D in a one-hybrid system devoid of retinoid X receptors. In addition, both Gal4-based systems behaved similar with regard to their dose-dependent response to vitamin D and related compounds when compared to the transcriptional activity of the vitamin D receptor in transiently transfected MCF-7 cells. Our results point out that specific ligands strongly enhanced receptor dimerization and induced transactivation in yeast and in MCF-7 cells. The constitutive transactivation by vitamin D receptor-retinoid X receptor heterodimers in yeast, depending on DNA binding of the receptors, strongly argues for the existence of cofactors, which are absent in yeast, but play a fundamental role in gene regulation in higher eukaryotic organisms.


Subject(s)
Receptors, Calcitriol/biosynthesis , Receptors, Calcitriol/genetics , Receptors, Retinoic Acid/biosynthesis , Receptors, Retinoic Acid/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcriptional Activation , Animals , Breast Neoplasms , Calcium-Binding Proteins/metabolism , Calreticulin , Dimerization , Humans , Ligands , Mice , Plasmids , Recombinant Fusion Proteins/metabolism , Retinoid X Receptors , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae/metabolism , Tumor Cells, Cultured
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