Involvement of SMRT corepressor in transcriptional repression by the vitamin D receptor.
To repress the expression of target genes, the unliganded nuclear receptor generally recruits the silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor via its direct association with the conserved motif within bipartite nuclear receptor-interaction domains (IDs) of the corepressor. Here, we investigated the involvement of the SMRT ... corepressor in transcriptional repression by the unliganded vitamin D receptor (VDR). Using small interference RNA against SMRT in human embryonic kidney 293 cells, we demonstrated that SMRT is involved in the repression of the VDR-target genes, osteocalcin and vitamin D(3) 24-hydroxylase in vivo. Consistent with this, VDR and SMRT are recruited to the vitamin D response element of the endogenous osteocalcin promoter in the absence of 1alpha,25-(OH)(2)D(3) in chromatin immunoprecipitation assays. To address the involvement of the VDR-specific interaction of SMRT in this repression, we identified the molecular determinants of the interaction between VDR and SMRT. Interestingly, VDR specifically interacts with ID1 of the SMRT/nuclear receptor corepressor and that ID1 is required for their stable interaction. We also identified specific residues in the SMRT-ID1 that are required for VDR binding, using the one- plus two-hybrid system, a novel genetic selection method for specific missense mutations that disrupt protein-protein interactions. These mutational studies revealed that VDR interaction requires a wide range of the residues within and outside the extended helix motif of SMRT-ID1. Notably, SMRT mutants defective in the VDR interaction were also defective in the repression of endogenous VDR-target genes, indicating that the SMRT corepressor is directly involved in the VDR-mediated repression in vivo via an ID1-specific interaction with the VDR.
Mesh Terms:
Amino Acid Sequence, Cell Line, DNA-Binding Proteins, Gene Expression Regulation, Humans, Inhibitor of Differentiation Protein 1, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Receptor Co-Repressor 2, Osteocalcin, RNA, Small Interfering, Receptors, Calcitriol, Repressor Proteins, Sequence Alignment, Steroid Hydroxylases, Transcription, Genetic, Two-Hybrid System Techniques
Amino Acid Sequence, Cell Line, DNA-Binding Proteins, Gene Expression Regulation, Humans, Inhibitor of Differentiation Protein 1, Molecular Sequence Data, Mutagenesis, Site-Directed, Nuclear Receptor Co-Repressor 2, Osteocalcin, RNA, Small Interfering, Receptors, Calcitriol, Repressor Proteins, Sequence Alignment, Steroid Hydroxylases, Transcription, Genetic, Two-Hybrid System Techniques
Mol. Endocrinol.
Date: Feb. 01, 2009
PubMed ID: 19098224
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