BIOCHEMISTRY OF LEUKEMIA VIRUS CORE BINDING FACTOR

核心结合因子(CBF)是一种异源二聚体转录因子 在造血中起中心作用的复合体。我们对CBF的兴趣 开始于我们发现CBF结合位点的一个突变 Moloney鼠白血病病毒增强子改变疾病特异性 Moloney MLV从T细胞淋巴瘤到红系白血病。我们提纯了 来自小牛胸腺的CBF，克隆了编码CBF复合体的cDNA，以及 证明了CBF由DNA结合亚单位(CBFpha)组成， 和一个非DNA结合亚基(CBFbeta)。编码两种基因的 CBFα和CBFβ亚基被染色体破坏 与人类急性白血病相关的易位。CBFA2 编码CBFAlpha亚单位的(AML1)基因被 急性髓系和淋巴细胞性T(8；21)、t(12：21)和t(3；21) 白血病，以及与治疗相关的白血病和骨髓发育不良。这个 Cbfb基因编码非DNA结合的CBFβ亚基，是 在急性髓系白血病中被inv(16)破坏。这些易位 导致嵌合蛋白的合成，这些嵌合蛋白保留了 结合到DNA中的CBF靶点，在那里他们可能放松了对 CBF靶基因的表达及其阻断分化的研究 造血细胞。CBFA2(AML1)和Cbfb基因加在一起是 在所有新发的急性白血病中，约有三分之一出现了中断， 使它们成为人类白血病中最常被破坏的基因。我们 通过突变Cbfa2确认CBF在造血中的重要性 和Cbfb基因，并证明这两种突变完全 扰乱体内的定向造血。我们还产生了胚胎 Cbfa2和Cbfb基因突变纯合的干细胞， 并表明这些ES细胞不能分化为 在体外或嵌合小鼠体内有明确的造血细胞。 这项提议的重点是与DNA结合的CBFalpha2亚单位。我们的整体 目的是描述CBFalpha2亚单位的功能结构域 及其致癌衍生物(特异靶1)，以确定三种 CBFalpha2 DNA结合域的空间结构(特定目的 2)，并表征细胞和分子基础 Cbfa2-1-小鼠的发育缺陷(特定目标3)。已被占用 总之，这些实验将提供详细的结构和 CBFalpha2蛋白功能结构域的生化信息 及其致癌衍生物，并将进一步加深我们对 CBF在正常发育和白血病中的作用

The core-binding factor (CBF) is a heterodimeric transcription factor complex that plays a central role in hematopoiesis. Our interest in CBF began with our discovery that a mutation in the CBF binding site in the Moloney murine leukemia virus enhancer changes the disease specificity of Moloney MLV from T cell lymphoma to erythroid leukemia. We purified CBF from calf thymus, cloned cDNAs encoding the CBF complex, and demonstrated that CBF is comprised of a DNA-binding subunit (CBFalpha), and a non-DNA-binding subunit (CBFbeta). Genes encoding both the CBFalpha and CBFbeta subunits are disrupted by chromosomal translocations associated with acute leukemias in humans. The CBFA2 (AML1) gene, which encodes a CBFalpha subunit, is disrupted by the t(8;21), t(12:21) and t(3;21) in acute myeloid and lymphocytic leukemias, and in therapy related leukemias and myelodysplasias. The CBFB gene, which encodes the non-DNA-binding CBFbeta subunit, is disrupted in acute myeloid leukemias by inv(16). These translocations result in the synthesis of chimeric proteins that retain the ability to bind to CBF target sites in DNA, where presumably they deregulate the expression of CBF target genes and block differentiation of hematopoietic cells. Together, the CBFA2 (AML1) and CBFB genes are disrupted in approximately one third of all de novo acute leukemias, making them the most frequently disrupted genes in human leukemias. We confirmed the importance of CBF in hematopoiesis by mutating the Cbfa2 and Cbfb genes in mice, and demonstrating that both mutations completely disrupt definitive hematopoiesis in vivo. We also generated embryonic stem (ES) cells homozygous for mutations in the Cbfa2 and Cbfb genes, and showed that these ES cells are incapable of differentiating into definitive hematopoietic cells either in vitro, or in chimeric mice. This proposal focuses on the DNA-binding CBFalpha2 subunit. Our overall goals are to characterize functional domains in the CBFalpha2 subunit and its oncogenic derivatives (Specific Aim 1), to determine the three dimensional structure of the CBFalpha2 DNA-binding domain (Specific Aim 2), and to characterize the cellular and molecular basis for the developmental defects seen in Cbfa2-1-mice (Specific Aim 3). Taken together, these experiments will provide detailed structural and biochemical information on functional domains of the CBFalpha2 protein and its oncogenic derivatives, and will further our understanding of the role of CBF in normal development and leukemia.