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 在造血中起核心作用的复合体。 我们对CBF的兴趣 开始于我们的发现，在CBF结合位点的突变， 莫洛尼鼠白血病病毒增强子改变疾病特异性 从T细胞淋巴瘤到红系白血病的莫洛尼MLV。 进行纯化 来自小牛胸腺的CBF，编码CBF复合物的克隆cDNA，和 证明CBF由DNA结合亚基（CBF α）组成， 和非DNA结合亚基（CBF β）。 基因编码两种 CBF α和CBF β亚基被染色体 与人类急性白血病相关的易位。 CBFA2 编码CBF α亚基的AML 1基因被 t（8;21）、t（12：21）和t（3;21）在急性髓系和淋巴细胞白血病中的表达 白血病，以及治疗相关的白血病和骨髓增生异常。 的 CBFB基因编码非DNA结合的CBF β亚基， 在急性髓性白血病中被inv破坏（16）。 这些易位 导致嵌合蛋白质的合成， 结合到DNA中的CBF靶位点，在那里它们可能会解除对CBF的调节。 CBF靶基因的表达和阻断分化 造血细胞 CBFA 2（AML 1）和CBFB基因共同作用， 在大约三分之一的新生急性白血病中被破坏， 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Cbfa 2证实了CBF在造血中的重要性， 和Cbfb基因，并证明这两种突变完全 破坏体内造血功能。我们还生成了胚胎 Cbfa 2和Cbfb基因突变纯合的干（ES）细胞， 并表明这些胚胎干细胞不能分化成 在体外或嵌合小鼠中的永久造血细胞。 该建议集中在DNA结合CBFalpha 2亚基上。 我们的整体 目的是表征CBFalpha 2亚基的功能结构域 及其致癌衍生物（特定目标1），以确定三个 CBFalpha 2 DNA结合结构域的三维结构（特异性目的 2），并表征细胞和分子基础， 在Cbfa 2 -1-小鼠中观察到的发育缺陷（具体目标3）。 采取 总之，这些实验将提供详细的结构和 CBFalpha 2蛋白功能结构域的生化信息 及其致癌衍生物，并将进一步我们的理解， 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.