AML1 IN NORMAL AND LEUKEMIC CELLS

AML-1/CBFβ转录因子复合体是最常见的 人类白血病易位的靶点，并在三分之一发生改变 急性髓系白血病和淋巴母细胞白血病。AML-1与 增强子核心DNA序列，被认为在 多种髓系和T细胞的谱系特异性表达 基因。为了研究AML-1的正常功能，我们生成AML-1- 通过同源重组获得缺陷小鼠。我们的初步结果 证明AML1-/-胚胎具有正常的形态发生和卵黄囊 源于造血，但完全没有胎肝 造血，并在胚胎中期发育过程中死于出血。 这些数据表明AML-1/CBFβ在调节 对最终的造血至关重要的基因的转录。 我们的假设是白血病相关的这种复合体的改变 导致AML-1介导的信号中断并导致异常 造血发育和最终的白血病。在这个项目中，我们有 提出了一系列实验来研究细胞的正常功能 AML-1，并确定t(8；21)编码的AML-1/ETO的效果 关于这些功能的产品。首先，我们将定义缺陷在哪里 由AML-1的丢失导致的在细胞水平上存在于 造血发育等级。这将涉及一项评估 AML1-/-卵黄囊祖细胞的造血活性 胚胎和AML1-/-分化潜能的测定 ES细胞，无论是在嵌合小鼠体内，还是在体外使用 类胚体和两步造血祖细胞集落分析。接下来，我们将 应用Cre-loxP-1研究AML-1在成人造血中的作用 AML1在出生后发育中的介导性选择性基因打靶 最后，我们将研究t(8；21)编码的AML-1/ETO的效果 正常造血的嵌合产物。这将包括一项分析 AML-1/ETO修复损失造成的缺陷的能力 AML-1和AML-1/ETO生殖系传播的后果。 总而言之，这些研究将提供对常态的有价值的见解 AML-1的功能，并有助于阐明这些基因的干扰 功能导致白血病的发生。

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.