A two-hybrid system for transactivator bait proteins

Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8726-31. doi: 10.1073/pnas.141413598. Epub 2001 Jul 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy-Related Proteins
  • Binding Sites
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Co-Repressor Proteins
  • Cyclin-Dependent Kinase 8
  • Cyclin-Dependent Kinases / genetics
  • Cyclin-Dependent Kinases / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Drosophila Proteins*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Gene Expression Regulation, Fungal
  • Helix-Loop-Helix Motifs
  • Histone Deacetylases
  • Humans
  • Insect Proteins / genetics
  • Insect Proteins / metabolism
  • Mice
  • MyoD Protein / genetics
  • MyoD Protein / metabolism
  • Nuclear Proteins*
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • RNA-Binding Proteins
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • TCF Transcription Factors
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Transcription Factor 7-Like 1 Protein
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic
  • Tumor Cells, Cultured
  • Two-Hybrid System Techniques

Substances

  • ATG16 protein, S cerevisiae
  • Autophagy-Related Proteins
  • Carrier Proteins
  • Co-Repressor Proteins
  • DNA-Binding Proteins
  • Drosophila Proteins
  • Fungal Proteins
  • GAL4 protein, S cerevisiae
  • GAL80 protein, S cerevisiae
  • Insect Proteins
  • MyoD Protein
  • Nuclear Proteins
  • Proto-Oncogene Proteins c-myc
  • RNA-Binding Proteins
  • Repressor Proteins
  • SAP18 protein, human
  • Saccharomyces cerevisiae Proteins
  • Sap18 protein, mouse
  • TCF Transcription Factors
  • TCF7L1 protein, human
  • TUP1 protein, S cerevisiae
  • Tcf7l1 protein, mouse
  • Trans-Activators
  • Transcription Factor 7-Like 1 Protein
  • Transcription Factors
  • Cyclin-Dependent Kinase 8
  • Cyclin-Dependent Kinases
  • SSN3 protein, S cerevisiae
  • Histone Deacetylases