ACTIVATION OF THE C-MYB PROTO-ONCOGENE

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The overall goal of this project is to understand the molecular mechanisms by which the c-myb proto-oncogene regulates normal and malignant hematopoiesis. We have recently shown that constitutive overexpression of the c-Myb protein is not sufficient to transform normal hematopoietic cells. In contrast, truncation of either the amino (N-) or the carboxyl (C-) terminus of c-Myb results in the transformation of promyelocytes (granulocyte precursors), whereas simultaneous truncation of both the N- and C-termini of c-Myb results in the transformation of myeloblasts (promyelocyte precursors). Both of these phenotypes differ from that of the monoblasts (macrophage precursors) transformed by the doubly truncated v-Myb protein of the avian myeloblastosis virus that contains eleven amino acid substitutions relative to c-Myb. We have also shown that the fusion of either the N- or C- terminus of c-Myb to the v-Myb protein suppresses transformation. These results imply that both the N- and C-termini of c- Myb can act as negative regulatory domains. The ability of different forms of c-Myb to transform different types of cells also suggests that c-Myb itself may participate in the control of hematopoietic lineage determination. This hypothesis is supported by the recent demonstration that mice with a homozygous disruption of c-myb are defective in fetal hematopoiesis. Accordingly, our specific aims for the next five years are as follows: (l) To precisely define which structural features of the N- and C-termini of c-Myb must be altered to cause transformation and alter lineage determination. (2) To determine the effects of alterations of the N- and C-termini of c- Myb on protein conformation, DNA-binding, and transcriptional regulation. (3) To determine the effects of alternatively spliced exons on the function of normal and altered forms o c-Myb. (4) To use high frequency homologous recombination in clonal chicken B cell lines to directly examine the role of c-Myb in hematopoietic lineage determination. (5) To analyze hematopoietic and lymphoid differentiation in transgenic lines of mice that express various forms of c-Myb, including the tissue- specific expression of dominant inhibitors of Myb function.