Point Mutations in Two EVI1 Zn Fingers Abolish EVI1-GATA1 Interaction and Allow Erythroid Differentiation of Murine Bone Marrow Cells

Abstract

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.

Ecotopic viral integration site 1 (EVI1) regulates multiple cellular processes important for cancer and is a synergistic partner for FOS protein in invasive tumors

“…This scan revealed significant (P < 0.0001) occurrence of 17 TF DNA binding motifs (Table S1). Two TFs previously reported as EVI1 interacting partners, GATA1 (27) and PU.1 (SPI1) (28), were among the overrepresented motifs. When we looked at the sequences of these 17 overrepresented TF motifs, we noticed that several were related to the EVI1 GATA or ETS-like motifs (Table S1).…”

“…S3A). Although these data do not exclude the possibility that these co-occurrences are functionally important (i.e., GATA1 is a reported EVI1 interacting partner that shares a nearly identical DNA binding motif) (27), we decided to focus on the 10 TF motifs that failed to bind EVI1 directly. These TF motifs include AP1 TRE, PAX2, GABPA, TEAD, ELK1, MYF, CTCF, YY1, ELK4, and ARNT.…”

EVI1 overexpression in distinct subtypes of pediatric acute myeloid leukemia

“…Other in vitro studies show that EVI1 overexpression leads to impaired erythroid and megakaryocytic differentiation by GATA-1 inactivation. [44][45][46] However, in vivo, no abnormalities of erythroid cells were observed in Evi1 transgenic mice, although they did show a significant reduction in the number of erythroid colony-forming units, implying a defect of erythroid hematopoiesis affecting erythroid progenitor cells. 47 Therefore, overexpression of EVI1 might be involved in the development of AML of both acute erythroid and megakaryoblastic leukemia.…”

Significant increase of self-renewal in hematopoietic cells after forced expression of EVI1

“…Phoenix retroviral packaging cells (ATCC) were maintained and transfected as previously described [9]. The viral supernatant was collected from the vector-or EVI1-transfected Phoenix cells, filtered and used for BM progenitors infection.…”