Interactions among vertebrate helix-loop-helix proteins in yeast using the two-hybrid system.
The helix-loop-helix (HLH) motif is contained in a rapidly growing family of transcription factors and has been shown to mediate dimerization among heterologous HLH-containing proteins. E12 is a widely expressed HLH protein that preferentially forms heterodimers with cell type-specific HLH proteins such as MyoD, myogenin, and the achaete-scute gene products. As a first step toward screening for novel cell type-specific partners of E12, we used a modification of the two-hybrid assay for detection of protein-protein interactions in vivo to determine whether dimerization of HLH proteins with E12 can occur in yeast. Using the GAL4 DNA-binding domain fused to the E12 HLH motif and the GAL4 transcription activation domain fused to MyoD, we show that E12 and MyoD can efficiently dimerize in yeast and reconstruct a hybrid transcription factor that activates reporter genes linked to the GAL4 DNA-binding site. The GAL4 DNA-binding domain fused to E12 was used to screen a mouse T-cell cDNA library in which the cDNA was fused to the GAL4 activation domain. Several cDNA clones encoding proteins that interact with E12 were isolated, one of which corresponded to the HLH protein Id-2. Given the ability of E12 to dimerize preferentially with cell type-specific HLH proteins, this strategy should be useful for cloning novel partners for E12 from a variety of cell types.
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
J
J
Staudinger
M
M
Perry
S J
SJ
Elledge
E N
EN
Olson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D051797:Inhibitor of Differentiation Protein 2; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism; D017570:MyoD Protein; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013601:T-Lymphocytes / Q000378:metabolism; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C497452:Idb2 protein, mouse; D051797:Inhibitor of Differentiation Protein 2; D009124:Muscle Proteins; D017570:MyoD Protein; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D051820:TCF Transcription Factors; C544913:Tcf7l1 protein, mouse; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D004247:DNA