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Involvement of SMRT Corepressor in Transcriptional Repression by the Vitamin D Receptor

Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, South Korea

Address all correspondence and requests for reprints to: Dr. Young Chul Lee, Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, South Korea. E-mail: yclee{at}jnu.ac.kr.

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 D₃ 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 1,25-(OH)₂D₃ 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.

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Coregulators:   VDR