Improved understanding of the regulation of lineage-specific gene expression during myelopoiesis is needed to clarify the fundamental mechanisms controlling normal hematopoietic cell differentiation and to identify therapeutic strategies that might reverse the disordered growth and development characteristic of the human myeloid leukemias. Genes encoding plasma membrane glycoproteins, expressed by cells of the myeloid lineage at well-defined stages of differentiation, offer attractive models for studying transcriptional regulation of hematopoietic cell development. This proposal focuses on two such genes, those encoding gp150 (CD13) and gp67 (CD33), which are expressed be committed human myeloid cell progenitors, subsets of more mature granulocytic and monocytic cells, and malignant blast cells from most cases of human myeloid leukemia. Biologically active molecular clones of these genes will be used to identify and characterize the promoter/enhancer sequences responsible for their lineage-specific regulation. Transcriptional regulatory sequences will be inserted upstream of the chloramphenicol acetyltransferase (CAT) gene and tested for their ability to program the expression of this """"""""reporter gene"""""""" in myeloid versus lymphoid leukemic cell lines. The activity of defined regulatory sequences from the human genes or their mouse homologues will be tested in vivo by introducing retroviral vectors containing these sequences into mouse hematopoietic stem cells. The cloned human gp150 cDNA will be employed as a """"""""reporter"""""""" gene, and its lineage-specific expression will be measured by flow cytometry in mouse hematopoietic cells. In other experiments, regulatory sequences will be fused to v- rasH coding sequences to determine whether leukemogenesis can be specifically programmed in developing myeloid cells. Complementary experiments will identify and characterize nuclear proteins that specifically interact with defined regulatory sequence elements to influence gene expression by myeloid cells. The proteins will be isolated according to their DNA-binding properties; specific antisera will be prepared; the genes that encode these hematopoietic cells will be examined. The studies outlined in this proposal will provide insights into the molecular basis of differential gene expression during hematopoiesis, and may suggest new approaches to the treatment of human myeloid leukemias.
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