Homotypic interactions of chicken GATA-1 can mediate transcriptional activation.
We used a one-hybrid system to replace precisely the finger II chicken GATA-1 DNA-binding domain with the binding domain of bacterial repressor protein LexA. The LexA DNA-binding domain lacks amino acids that function for transcriptional activation, nuclear localization, or protein dimerization. This allowed us to analyze activities of GATA-1 sequences distinct from DNA binding. We found that strong transcriptional activating sequences that function independently of finger II are present in GATA-1. Sequences including finger I contain an independent nuclear localizing function. Our data are consistent with cooperative binding of two LexA-GATA-1 hybrid proteins on a palindromic operator. The sensitivity of our transcription assay provides the first evidence that GATA-1 can make homotypic interactions in vivo. The ability of a non-DNA-binding form of GATA-1 to activate gene expression by targeting to a bound GATA-1 derivative further supports the notion that GATA-1-GATA-1 interactions may have functional consequences. A coimmunoprecipitation assay was used to demonstrate that GATA-1 multimeric complexes form in solution by protein-protein interaction. The novel ability of GATA-1 to interact homotypically may be important for the formation of higher-order structures among distant regulatory elements that share binding sites for this transcription factor. We also used the system to test the ability of GATA-1 to interact heterotypically with other activators.
Yang
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Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Evans
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D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002645:Chickens; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D017354:Point Mutation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein