Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells.
EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.
Laricchia-Robbio
Leopoldo
L
University of Illinois at Chicago (M/C 737), 909 South Wolcott Avenue, Chicago, IL 60612, USA.
Fazzina
Raffaella
R
Li
Donglan
D
Rinaldi
Ciro R
CR
Sinha
Kisaly K
KK
Chakraborty
Soumen
S
Nucifora
Giuseppina
G
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D041905:Erythroid Cells / Q000166:cytology* / Q000378:metabolism*; D050982:GATA1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D017354:Point Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D050982:GATA1 Transcription Factor; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins