In vitro transcription of erythroid promoters using baculoviral-expressed human GATA-1: purification, physicochemistry, and activities.
GATA-1 is a cys-2/cys-2 zinc finger transcriptional activator that is required for erythrocyte development in chimeric mice and contributes to the expression of all erythroid genes studied to date, including the erythropoietin receptor, glycophorin B, and porphobilinogen deaminase genes. Transactivation by GATA-1 is mediated by either an amino-terminal acidic domain, R1, or an independent adjacent domain, R2, and may involve the coordinate action of cofactors (NF-E2, EKLF, and Sp1) which bind adjacent cis-elements. To directly assess mechanisms of transactivation, we have developed an efficient cell-free transcription system using recombinant human GATA-1 (rhGATA-1) expressed in SF9 cells. Levels of baculoviral expression of GATA-1 were > or = 200-fold higher than endogenous levels in erythroid K562 cells. Factors from each source were essentially equivalent in molecular weight and DNA binding properties, and highly similar in phosphotryptic peptide composition. Notably, DNA binding was inhibited following treatment with alkaline phosphatase. In both SF9 and K562 cells, GATA-1 occurred largely as heterogeneous multimers, thus complicating its isolation by standard procedures. However, significant purification of this factor (> or = 100-fold; > or = 75% purity) was accomplished via DNA affinity chromatography. In cell-free assays, this rhGATA-1 was shown to be remarkably active in transactivating model erythroid promoters. This work establishes an efficient in vitro system for direct analyses of mechanisms, cofactors, and functional domains of GATA-1 which regulate transcription at defined proximal promoters.
Department of Biochemistry & Molecular Biology, Pennsylvania State University, University Park 16802.
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Taxman
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Sonsteby
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Wojchowski
D016367:Baculoviridae / Q000378:metabolism; D001483:Base Sequence; D002474:Cell-Free System; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006801:Humans; D008969:Molecular Sequence Data; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D015534:Trans-Activators / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D015534:Trans-Activators; D014157:Transcription Factors