title: Identification and characterization of a novel corepressor interaction region in RVR and Rev-erbA alpha.
abstract: Rev-erbA alpha and RVR are orphan nuclear receptors that function as dominant transcriptional silencers. Ligand-independent repression of transcription by Rev-erbA alpha and RVR is mediated by the nuclear receptor corepressors, N-CoR and its variants RIP (RXR interacting protein) 13a and RIP13 delta 1. The physical association between the corepressors and Rev-erbA alpha and RVR is dependent on the presence of a receptor interaction domain (RID) in the N-CoR family. Our previous study demonstrated that the E region of RVR and Rev-erbA alpha is necessary and sufficient for the in vivo interaction with the nuclear receptor corepressor, RIP13 delta 1. The present investigation demonstrates that two corepressor interaction regions, CIR-1 and CIR-2, separated by approximately 150 amino acids in the E region of RVR, are required for the interaction with N-CoR, RIP13a, and RIP13 delta A. The D region is not required for the physical interaction. In contrast, the D and E regions of Rev-erbA alpha were necessary for the interaction with the N-CoR and RIP13a-RIDs in vivo, suggesting that RIP13 delta 1 and N-CoR/RIP13a differentially interact with Rev-erbA alpha. Mutagenesis of CIR-1, a novel domain that is highly conserved between RVR and Rev-erbA alpha, demonstrated that the N-terminal portion of helix 3 plays a key role and is absolutely necessary for the interaction with RIP13 delta 1, RIP13a, and N-CoR. The phenylalanine residues, F402 and F441, in RVR and Rev-erbA alpha, respectively, were critical residues in supporting corepressor interaction. Cotransfection studies demonstrated that repression of a physiological target, the human Rev-erbA alpha promoter, by RVR was significantly impaired by mutation of CIR-1 or deletion of CIR-2. Furthermore, overexpression of either the N-CoR/RIP13a or RIP13 delta 1-RIDs alleviated RVR-mediated repression of the Rev-erbA alpha promoter, demonstrating that corepressor binding mediates the repression of a native target gene by RVR. A minimal region containing juxtapositioned CIR-1 and CIR-2 was sufficient for corepressor binding and transcriptional repression. In conclusion, our study has identified a new corepressor interaction region, CIR-1, in the N terminus of helix 3 in the E region of RVR and Rev-erbA alpha, that is required for transcriptional silencing. Furthermore, we provide evidence that CIR-1 and CIR-2 may form a single corepressor interaction interface.
authors.affiliation: University of Queensland, Centre for Molecular and Cellular Biology, Ritchie Research Laboratories, Brisbane, Australia.
authors.forename: L J
authors.initials: LJ
authors.lastname: Burke
authors.forename: M
authors.initials: M
authors.lastname: Downes
authors.forename: V
authors.initials: V
authors.lastname: Laudet
authors.forename: G E
authors.initials: GE
authors.lastname: Muscat
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002822:Choriocarcinoma; D004268:DNA-Binding Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D056971:Nuclear Receptor Co-Repressor 1; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; C539127:NCOR1 protein, human; C064353:NR1D1 protein, human; C539128:Ncor1 protein, mouse; D009419:Nerve Tissue Proteins; C498262:Nr1d1 protein, mouse; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011988:Receptors, Thyroid Hormone; D012097:Repressor Proteins