The use of altered specificity mutants to probe specific protein-protein interactions involved in the activation of GATA-1 target genes.
With the increasing popularity of the yeast two-hybrid screen, a large number of protein-protein interactions have been identified. In many cases, single proteins have been found to associate with a large number of cofactors. For example, the hematopoietic transcription factor GATA-1 interacts with a multitude of other nuclear proteins, including Friend of GATA-1 (FOG-1), EKLF, CBP/p300, and Lmo2. Furthermore, p300, besides associating with GATA-1, interacts with at least seven other hematopoietic transcription factors. Despite the numerous pairwise and higher-order interactions identified, assessment of their contribution to transcriptional control has been lacking. Here we describe a strategy that can be applied to assess the functional relevance of any protein-protein association. This approach involves the creation of altered specificity mutants though the use of a combination of two yeast two-hybrid screens. Once altered specificity factors are obtained, a researcher can then proceed to functional assays that address the role of a specific protein-protein interaction.
Crispino
John D
JD
Ben May Institute for Cancer Research, University of Chicago, IL 60637, USA. jcrispin@huggins.bsd.uchicago.edu
Orkin
Stuart H
SH
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D005786:Gene Expression Regulation; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; C498432:ZFPM1 protein, human