The long-term goal of this project is to understand how the c-Myb and v-Myb proteins are regulated, and how they participate in transformation and oncogenesis. The c-Myb protein is a transcription factor that is required for hematopoiesis and that has been implicated in the regulation of cell proliferation, differentiation and apoptosis. Although it is expressed in a variety of myeloid, lymphoid and epithelial malignancies, c-Myb overexpression does not induce tumors because the protein is subject to inherent negative regulation. In contrast, v-Myb is an activated allele of c-Myb expressed by Avian Myeloblastosis Virus that readily transforms hematopoietic cells in vitro and induces leukemia an animals. The v-Myb protein has been modified by truncations and point mutations that disrupt protein binding sites involved in negative regulation. The three Specific Aims proposed here outline a combined biochemical, molecular and genetic approach to answer the question: How do protein interactions with the Myb DNA binding domain regulate its activity? In Specific Aim number 1, the individual binding sites for important regulatory proteins will be mapped in the Myb DNA binding domain. Then, Myb proteins carrying binding site mutations will be tested in a variety of functional assays, to test the importance of Myb-regulator interactions in vivo.
In Specific Aim number 2, dominant negative versions of two Myb-binding regulatory proteins will be used to control c-Myb or v-Myb activity. By expressing regulatable or inducible versions of these dominant negative proteins in cells that express c-Myb, the importance of the regulatory proteins on downstream Myb-dependent functions will be assessed. Finally, in Specific Aim number 3, the importance of acquired mutations in c-Myb proteins expressed by human tumors and cell lines will be addressed. Most importantly, the functional significance of any mutations will be tested using assays developed in the Principal Investigator's laboratory. The proposed experiments will shed light on the regulation of hematopoietic cell differentiation, apoptosis and proliferation as well as the molecular events leading to leukemias and other Myb-expressing tumors. These experiments are logical extensions of earlier work in the Principal Investigator's laboratory, and take advantage of the experimental tools, assay systems and expertise developed during the previous project period.
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