Protein-protein interaction between Fli-1 and GATA-1 mediates synergistic expression of megakaryocyte-specific genes through cooperative DNA binding.
Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each other's activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbalpha in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIbalpha genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.
Eisbacher
Michael
M
Centre for Thrombosis and Vascular Research, School of Medical Sciences and Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia.
Holmes
Melissa L
ML
Newton
Anthea
A
Hogg
Philip J
PJ
Khachigian
Levon M
LM
Crossley
Merlin
M
Chong
Beng H
BH
D020816:Amino Acid Motifs; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002469:Cell Separation; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D005434:Flow Cytometry; D050982:GATA1 Transcription Factor; D015723:Gene Library; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D020014:K562 Cells; D007700:Kinetics; D008533:Megakaryocytes / Q000378:metabolism*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers; D000099232:Proto-Oncogene Protein Spi-1
D004247:DNA; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005982:Glutathione Transferase; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D012313:RNA; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C494688:GATA1 protein, human