Abstract
EVI1 is a nuclear oncoprotein deregulated by recurring chromosomal abnormalities in MDS. The expression of this gene in MDS patients represents a very poor prognostic marker and is associated with erythroid and megakaryocytic defects. We have previously shown that the forced expression of EVI1 in mice results in a fatal disease with features characteristic of MDS including dyserythropoiesis, dysmegakaryopoiesis, and anemia. More recently we have shown that EVI1 directly interacts with GATA1 and disrupts GATA1−binding to DNA leading to deregulation of GATA1−dependent genes.
Here we describe the effects of an EVI1 mutant unable to bind GATA1, on the regulation of GATA1 target genes and on the erythroid differentiation of murine bone marrow progenitors. The structure of two zinc finger motifs of this mutant, EVI1(1+6Mut), were destroyed by His to Ala and Cys to Ala changes. Semi−quantitative RT−PCR showed that most of the analysed genes were down−regulated in 32Dcl3 cells expressing EVI1 but not EVI1(1+6Mut). Bone marrow lineage negative cells infected with EVI1, EVI1(1+6Mut), or the empty vector as control, selected in G418, and plated in presence of Epo were utilized to determine the progenitors’ potential to differentiate in response to this cytokine. The EVI1−expressing cells were virtually unable to generate erythroid colonies after Epo stimulation and only scarce small colonies were observed. In contrast, the EVI1(1+6Mut)−cells produced about 65% of the colonies formed by the control cells. The cells were recovered and their morphology was analysed after Wright−Giemsa staining. All EVI1−positive erythroid cells showed an impaired differentiation that was arrested at the basophilic−erythroblast stage. These cells were bi− and tetra−nucleated with chromatin bridges, budding nuclei and nuclear−cytoplasmatic maturative asynchronizations. All of these features are known as dysplastic erythroid aspects in MDS patients. These dysplastic characteristics were less prominent in EVI1(1+6Mut)−positive cells and were observed only in a minority of cells rather than in the entire population. As expected, the control cells had the appearance of normal erythroblasts and none of the aberrant features were observed. These results parallel the morphology observation of peripheral blood smears obtained from EVI1−mice and EVI1(1+6Mut)−mice in that erythropoiesis defects such as aniso−poikilocytosis and orthochromatic erythroblasts were less striking and observed only in a limited number of erythroid cells in EVI1(1+6Mut)−mice. Based on these results, we propose that EVI1 blocks erythroid differentiation by direct interaction with GATA1 leading to impairment of GATA1 regulation. This differentiation block is significantly reduced in vitro and in vivo with the EVI1(1+6Mut) mutant, which re−establishes to a high degree the normal functions of GATA1 and diminishes the erythroid dysplastic aspects observed in EVI1 cells.
Changes in the lactate dehydrogenase isoenzyme pattern during differentiation of rabbit bone-marrow erythroid cells
