AML1 IN NORMAL AND LEUKEMIC CELLS

AML-1/CBFbeta转录因子复合物是最常见的 在人类白血病易位的目标，并改变了三分之一， 急性骨髓性和淋巴细胞性白血病 AML-1结合于 增强子核心DNA序列，并被认为在增强子核心DNA序列中起关键作用。 一些骨髓和T细胞特异性的谱系特异性表达 基因. 为了研究AML-1的正常功能，我们产生了AML-1， 缺陷小鼠通过同源重组。 我们的初步结果 表明AML 1-/-胚胎具有正常的形态发生和卵黄囊- 衍生造血，但完全没有胎肝 在胚胎中期发育期间死于发育不良。 这些数据表明，AML-1/CBF β在调节 基因的转录是决定性造血所必需的。 我们的假设是白血病相关的这种复合物的改变 导致AML-1介导信号破坏并导致异常的 造血发育和最终的白血病。 在这个项目中， 提出了一系列的实验，以调查正常的功能， AML-1，并确定t（8;21）编码的AML-1/ETO的作用 这些功能的产品。 首先，我们将定义缺陷 由AML-1缺失引起的白血病的发生在细胞水平， 造血发育层次 这将涉及评估 AML 1-/-卵黄囊祖细胞的造血活性 胚胎，并测定AML 1-/- ES细胞，在嵌合小鼠体内和体外使用 拟胚体和两步造血集落测定。我们接下来将 用Cre-loxP-2研究AML-1在成人造血中的作用。 在出生后发育中介导的选择性基因靶向AML 1。 最后，我们将研究t（8;21）编码的AML-1/ETO的影响 嵌合产物对正常造血的影响。 这将包括分析 AML-1/ETO修复损失造成的缺陷的能力 的AML-1，和AML-1/ETO的生殖系传播的后果。 这些研究将为正常的 AML-1的功能，并帮助阐明如何破坏这些功能， 功能导致白血病发生。

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.