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.
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