The AML-1/CBFbeta transcription factor complex is the most frequent target of translocations in human leukemia and is altered in one-third of acute myeloid and lymphoblastic leukemias. AML-1 binds to the enhancer core DNA sequence and is believed to play a critical role in the lineage-specific expression of a number of myeloid and T-cell specific genes. To investigate the normal function of AML-1, we generate AML-1- deficient mice by homologous recombination. Our preliminary results demonstrate that AML1-/- embryos have normal morphogenesis and yolk sac- derived hematopoiesis, but have a complete absence of fetal liver hematopoiesis, and die from hemorrhages during mid-embryonic development. These data suggest that AML-1/CBFbeta plays a pivotal role in regulating transcription of genes that are essential for definitive hematopoiesis. Our hypothesis is that leukemia-associated alterations of this complex lead to disruption of AML-1-mediated signals and result in abnormal hematopoietic development and eventual leukemia. In this project we have proposed a series of experiments to investigate the normal functions of AML-1, and to determine the effects of the t(8;21)-encoded AML-1/ETO products on these functions. First we will define where the defect resulting from the loss of AML-1 lies at a cellular level within the hematopoietic developmental hierarchy. This will involve an evaluation of the hematopoietic activity of yolk sacs progenitors from AML1-/- embryos, and determination of the differentiation potential of AML1-/- ES cells, both in vivo in chimeric mice, and in vitro using cultures of embryoid bodies and two-step hematopoietic colony assays. We will next investigate the role of AML-1 in adult hematopoiesis by use of Cre-loxP- mediated selective gene targeting of AML1 in postnatal development. Lastly, we will investigate the effects of the t(8;21)-encoded AML-1/ETO chimeric product on normal hematopoiesis. This will include an analysis of the ability of AML-1/ETO to rescue the defects resulting from the loss of AML-1, and of the consequences of germline transmission of AML-1/ETO. Together these studies will provide valuable insights into the normal functions of AML-1 and help to elucidate how disruption of these functions leads to leukemogenesis.
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