BIOCHEMISTRY OF LEUKEMIA VIRUS CORE BINDING FACTOR

核心结合因子（CBF）是一种异二聚体转录因子 在造血过程中发挥核心作用的复合物。 我们对 CBF 的兴趣 首先我们发现 CBF 结合位点发生突变 莫洛尼鼠白血病病毒增强剂改变疾病特异性 Moloney MLV 从 T 细胞淋巴瘤到红细胞白血病。 我们净化了 来自小牛胸腺的 CBF，编码 CBF 复合物的克隆 cDNA，以及 证明 CBF 由 DNA 结合亚基 (CBFalpha) 组成， 和非 DNA 结合亚基 (CBFbeta)。 编码这两种基因的基因 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 (AML1) 基因编码 CBFα 亚基，被 急性髓细胞和淋巴细胞中的 t(8;21)、t(12:21) 和 t(3;21) 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 CBFbeta 亚基， 在急性髓性白血病中，该基因被 inv(16) 破坏。 这些易位 导致嵌合蛋白的合成，该蛋白保留了 与 DNA 中的 CBF 靶位点结合，推测它们会解除对 CBF 的调节 CBF靶基因的表达和块分化 造血细胞。 CBFA2 (AML1) 和 CBFB 基因一起 大约三分之一的新发急性白血病被破坏， 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 Cbfa2 证实了 CBF 在造血中的重要性 和 Cbfb 基因在小鼠中，并证明这两种突变完全 破坏体内确定的造血作用。我们还产生了胚胎 Cbfa2 和 Cbfb 基因突变纯合的干细胞 (ES)， 并表明这些 ES 细胞无法分化为 体外或嵌合小鼠中的定形造血细胞。 该提案重点关注 DNA 结合 CBFalpha2 亚基。 我们的整体 目标是表征 CBFalpha2 亚基的功能域 及其致癌衍生物（具体目标 1），以确定三个 CBFalpha2 DNA 结合域的维度结构（具体目标 2) 并表征其细胞和分子基础 Cbfa2-1-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 CBFalpha2 蛋白功能域的生化信息 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.