ABSTRACT
Nuclear receptor corepressor (N-CoR) regulates gene expression through interaction with DNA-bound nuclear receptors, recruiting multicomponent repressor complexes to the sites of target genes. We recently reported the presence of an LXXLL motif in N-CoR, and showed that this motif interacts in vitro and in vivo with retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta). Transient transfection experiments now suggest that TRbeta and N-CoR act synergistically and may both be required for ligand-induced repression from the negative TR response element in the thyroid stimulating hormone-beta (TSHbeta) gene promoter. Mutation of the LXXLL motif in N-CoR abolished ligand-induced repression at this response element. Furthermore, in vitro binding of N-CoR to a complex between TRbeta and the negative TR response element was strictly ligand-dependent. We conclude that N-CoR and TRbeta cooperate in the regulation of the TSHbeta gene and that the ligand-dependent repression is mediated by the LXXLL motif in N-CoR.
Subject(s)
Thyrotropin, beta Subunit/chemistry , Amino Acid Motifs , Animals , Blotting, Western , Cell Line , DNA/chemistry , Drosophila , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Glutathione/metabolism , Histone Acetyltransferases/chemistry , Humans , Ligands , Mice , Mutation , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , RNA, Small Interfering/metabolism , Receptors, Retinoic Acid/metabolism , Recombinant Proteins/chemistry , Response Elements , Retinoic Acid Receptor alpha , Time Factors , Transcription, Genetic , TransfectionABSTRACT
Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis. Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor beta and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively. Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors. N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man. In contrast, SMRT lacks this motif. This paper describes functional implications of the LXXLL motif in N-CoR. A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoR(LDNLL)) fused to GST interacted with retinoic acid receptor alpha (RARalpha) and thyroid hormone receptor beta (TRbeta) in vitro. Similarly, [(35)S-methionine]N-CoR(LDNLL) interacted with a RARalpha fusion protein. N-CoR(LDNLL) also bound to RARalpha in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria. The interaction with RARalpha in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA. Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RARalpha and TRbeta.
Subject(s)
DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Receptors, Retinoic Acid/physiology , Receptors, Thyroid Hormone/physiology , Repressor Proteins/physiology , Transcription Factors/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , Cells, Cultured , Conserved Sequence , Gene Expression Regulation , Histone Acetyltransferases , Humans , Ligands , Mice , Molecular Sequence Data , Mutation , Nuclear Receptor Co-Repressor 1 , Nuclear Receptor Co-Repressor 2 , Nuclear Receptor Coactivator 1 , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Retinoic Acid Receptor alpha , Sequence Homology, Amino Acid , Thyroid Hormone Receptors beta , Trans-Activators/physiology , Two-Hybrid System TechniquesABSTRACT
Variation in cell morphology and function is caused by differentiation. In myeloid differentiation, retinoid signaling, acting through heterodimers consisting of retinoic acid receptor and retinoid X receptor (RAR/RXR) plays a crucial part. The RAR/RXR heterodimers bind to naturally occurring response elements in the promoter regions of target genes, deciding whether the gene is to be transcribed or not. In the absence of the RAR-specific ligand all trans retinoic acid, RAR/RXR heterodimers are associated with the nuclear receptor corepressor N-CoR or the related SMRT. Here we show, using Western, far-Western and Northern blot techniques, that when the human monocytic cell line THP-1 is allowed to differentiate into macrophage-like cells the expression of N-CoR is down-regulated both at the protein and at the mRNA level. To investigate how this affects the transcriptional activity of retinoic acid response element (RARE)-controlled genes, we performed transient transfection experiments in THP-1 and CV-1 cells. The results indicate that N-CoR functions not merely as a repressor of basal transcription, but rather as a modulator of both basal and ligand-activated transcription of genes controlled by RAR/RXR heterodimers in a dose-dependent manner.
