The long term objectives of this application are to understand how hematopoietic specific transcription factors regulate gene expression resulting either in proper blood cell development or leukemia. Studies are focused on the protein PU.1, which is normally found in B lymphocytes and myeloid lineages. Knockout mice have shown that PU.1 is required for the development and function of multiple blood cell lineages. In contrast, integration of the SFFV virus into the PU.1 locus has shown that over-expression of PU.1 in erythroblasts results in erythroleukemia in mice. Thus, PU.1 is a transcription factor and an oncogene whose expression controls normal versus abnormal blood cell development. The studies outlined in this proposal are divided into two specific aims. In the first specific aim, the applicant will study the role of PU.1 in four inter-related models of normal and abnormal blood cell development. In the first model, he will re-express PU.1 in ES-/- null cells and study their differentiation into myeloid cells to study the role of PU.1 in normal development. He will also use his unique collection of mutant PU.1 genes to correlate biological activity with the known functional domains. In the second model, he will transduce murine bone marrow cells with a PU.1 expressing retrovirus and transplant these cells back into irradiated syngeneic mice. He will study whether PU.1 can function as an oncogene in multiple cell types or is it restricted to the erythroid lineage. In the third model, he will target PU.1 overexpression to a defined cell lineage which does not normally express PU.1, T cells, and study the development and function of these cells. For the fourth model, he is collaborating on studies designed to show the functional role of PU.1 in the development of erythroleukemia. In the second specific aim, the applicant will use the techniques of differential display and representational difference analysis to clone genes whose expression is turned on or off as the result of PU.1 expression in these four models. Through these studies he will compare the sets of genes PU.1 is regulating in normal and abnormal hematopoiesis. Results from these studies will provide valuable information on how an individual transcription factor such as PU.1 can regulate decisions of gene expression. It is these decisions during lineage commitment and differentiation of blood cells that is the difference between normal blood cell development and leukemia.
