Oncogenic Transcription Factor Evi1 Regulates Hematopoietic Stem Cell Proliferation through GATA-2 Expression.

Chromosomal abnormalities, such as translocation, mutation or deletion, are central to the pathogenesis of human cancers. Recently, several transcription factors have been isolated as genes responsible for leukemia from the region surrounding chromosomal breakpoints, which are implicated in the regulation of normal hematopoiesis. Among on them, ecotropic viral integration site-1 (Evi1) is a transcription factor activated by retroviral integration in murine leukemias and chromosomal rearrangements in human leukemias. Evi1 is a zinc finger transcription factor and contains two separated DNA-binding domains. It was reported that Evi1−/− embryos die at approximately E10.5, exhibiting widespread hypocellularity and hemorrhaging. However, the role in normal hematopoiesis or authentic target genes of Evi1 has not been elucidated.

Here, we show that Evi1 is predominantly expressed in hematopoietic stem cells (HSCs) in embryos and adult bone marrows, and Evi1−/− embryos are markedly decreased in numbers of HSC. One embryo-equivalent cells from E9.5 P-Sp of Evi1+/+, Evi1+/− and Evi1−/− embryos (Ly5.2) were transplanted into a busulfan-conditioned newborn recipient (Ly5.1). At 2 months posttransplant, donor-derived Ly5.2(+) cells could be detected in the peripheral blood of the recipients that received P-Sp cells from the Evi1+/+ and Evi1+/− but not from the Evi1−/− embryos. Thus, Evi1 is critical for the generation of HSCs in the P-Sp. Both Evi1−/− embryos and yolk sac showed marked retardation in the organization of the vascular system, particularly in vascular remodeling, compared with controls. Using an in vitro P-Sp culture analysis, we found normal in vitro differentiation of endothelial cells in Evi1−/− P-Sp cultures but defects in their in vitro network formation, which is normally promoted by Ang-1 secreted from developing HSCs in P-Sp cultures. HSCs from adult bone marrow or HSCs from E9.5 wild type embryos rescued defective angiogenesis in Evi1−/− embryos. The fine vascular network coincided with the region where HSCs formed a colony. Their round morphology confirmed that exogenous adult HSCs did not differentiate into elongated endothelial cells. We showed that recombinant Ang-1 alone restored the defective angiogenesis in Evi1−/− embryos to a wild type level. It is suggested that the defect in hematopoietic cells induced defective angiogenesis in Evi1−/− embryos mediated by Ang-1. Notably, mRNA expression of GATA-2, which is essential for proliferation of definitive HSCs, was profoundly reduced in Evi1−/− embryos. Analysis of the GATA-2 promotor revealed that Evi1 directly binds to the 5′ upstream region of the GATA−2 exon and positively regulates its promoter activity in vitro and in vivo. Restoration of GATA-2 expression dramatically rescued the defective expansion of Evi1−/− embryos HSCs in vitro. Our results reveal that GATA-2 is a critical in vivo target for Evi1 and indicate hierarchical regulation of the HSC pool by transcriptional regulators.