Role of EVI1 in Cell Cycle Regulation: Relevance of Specific Target Genes.

Mds1-Evi1 is a complex locus with two distinct transcriptional start sites, four possible polyadenylation sites, and nineteen exons spread over half a megabase that encodes at least six protein isoforms containing variable numbers of DNA-binding zinc finger motifs. In mouse and some other species, the locus is a common site of proviral insertion in myeloid leukemia, while in human it is a site of nonrandom chromosomal rearrangement in myelodysplasia, AML, and CML blast crisis. One such translocation is t(3;21), encoding the AML1-MDS1-EVI1 (AME) fusion protein. We have characterized the RNA transcripts and proteins generated by Mds1-Evi1 in murine leukemic cells bearing Evi1 proviral insertion using three different antisera specific for the N-terminal and C-terminal domains of EVI1, as well as MDS1, and find consistent overexpression of three protein isoforms of 135, 123, and 103 kDa (EVI1A, B, and C, respectively) while other isoforms, including MDS1-EVI1, are not normally expressed. However, by knocking lacZ into the Mds1 first exon we showed that it is normally expressed in hematopoietic progenitors, similar to Evi1. The N-terminal zinc finger domain present in the EVI1A and B isoforms binds to the GACAAGATA motif with high affinity, and single missense mutations in zinc finger six abrogate high affinity binding. Using two different bioassays, we have demonstrated that cellular transformation by either the EVI1A isoform or by AME is dependent on DNA binding via this N-terminal domain, thus indicating that transformation is dependent on the action of this domain on specific target genes. To determine the biological effect of Evi1 on leukemic cell growth, short hairpin RNAs were used to suppress Evi1, and this resulted in slowing of the cell cycle and prolongation of the G2/M phase. RNA expression profiling revealed significant changes in gene expression accompanied Evi1 suppression, including some involved in cell cycle control. Upon long term Evi1 suppression, numerous markers of myeloid differentiation were induced, indicating the acquisition of a differentiation-like phenotype. These data indicate an effect of EVI1 on both cell cycle and differentiation, and suggest that the effect on cell cycle is direct. To gain further insight into the nature of EVI1 target genes, we utilized tet-regulated expression of Evi1A, and identified a set of putative targets of the A isoform that includes genes directly involved in cell cycle. However, the fold changes are relatively minor, and although the kinetics of activation suggests these are direct targets, this has not been conclusively demonstrated. We predict on the basis of these data that EVI1 expression modulates several key regulators of cell cycle and this results in an acceleration of cell cycle. With high level EVI1 expression, block to differentiation is induced as well.