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.
Kim
Ji Young
JY
School of Biological Sciences and Technology, Chonnam National University, Buk-gu, Gwangju 500-757, South Korea.
Son
You Lee
YL
Lee
Young Chul
YC
D000595:Amino Acid Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D056985:Nuclear Receptor Co-Repressor 2; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D065668:Vitamin D3 24-Hydroxylase
D004268:DNA-Binding Proteins; C497453:ID1 protein, human; D051798:Inhibitor of Differentiation Protein 1; C539130:NCOR2 protein, human; D056985:Nuclear Receptor Co-Repressor 2; D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol; D012097:Repressor Proteins; D015675:Osteocalcin; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase