title: A two-hybrid system for transactivator bait proteins.
abstract: We describe a two-hybrid strategy for detection of interactions with transactivator proteins. This repressed transactivator (RTA) system employs the N-terminal repression domain of the yeast general repressor TUP1. TUP1-GAL80 fusion proteins, when coexpressed with GAL4, are shown to inhibit transcription of GAL4-dependent reporter genes. This effect requires the C-terminal 30 residues of GAL4, which are required for interaction with GAL80 in vitro. Furthermore, repression of GAL transcription by TUP1-GAL80 requires SRB10, demonstrating that the TUP1 repression domain, in the context of a two-hybrid interaction, functions by the same mechanism as endogenous TUP1. Using this strategy, we demonstrate interactions between the mammalian basic helix-loop-helix proteins MyoD and E12, and between c-Myc and Bin-1. We have also identified interacting clones from a TUP1-cDNA fusion expression library by using GAL4-VP16 as a bait fusion. These results demonstrate that RTA is generally applicable for identifying and characterizing interactions with transactivator proteins in vivo.
authors.affiliation: Department of Biochemistry and Molecular Biology; University of British Columbia Vancouver, BC, Canada V6T 1Z3.
authors.forename: M
authors.initials: M
authors.lastname: Hirst
authors.forename: C
authors.initials: C
authors.lastname: Ho
authors.forename: L
authors.initials: L
authors.lastname: Sabourin
authors.forename: M
authors.initials: M
authors.lastname: Rudnicki
authors.forename: L
authors.initials: L
authors.lastname: Penn
authors.forename: I
authors.initials: I
authors.lastname: Sadowski
mesh_terms: D000818:Animals; D000071183:Autophagy-Related Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D056970:Co-Repressor Proteins; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D018257:Helix-Loop-Helix Motifs; D006655:Histone Deacetylases; D006801:Humans; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
chemical_list: C498649:ATG16 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D056970:Co-Repressor Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D019476:Insect Proteins; D017570:MyoD Protein; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C498650:SAP18 protein, human; D029701:Saccharomyces cerevisiae Proteins; C498651:Sap18 protein, mouse; D051820:TCF Transcription Factors; C544912:TCF7L1 protein, human; C066502:TUP1 protein, S cerevisiae; C544913:Tcf7l1 protein, mouse; D015534:Trans-Activators; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases; C498309:SSN3 protein, S cerevisiae; D006655:Histone Deacetylases