This application for a Mentored Clinical Scientist Development Award is envisaged to facilitate the candidate's transition from a post-doctoral fellow to an independent investigator. 90% of the effort will be devoted to conducting the experiments that seek to further identify the cis-regulatory elements of the GATA-1 transcription factor gene and understand their function. The research will be conducted in the Hematology Division of a major teaching hospital in a rich and supportive environment that has an established commitment to the professional development of physician-scientists. The remainder of the effort will be applied toward continued comprehensive clinical training in Medical Oncology and Hematology as a non-salaried Clinical Instructor of Medicine. GATA-1 is a lineage-restricted transcription factor that is required for blood formation. It is likely that identification of the cis-acting DAN regulatory elements of this gene and the corresponding factors that interact with these sequences will provide new insights into how hematopoietic cell lineage commitment and differentiation programs are established. In addition, these cis-elements will provide gene-cassettes available for a wide variety of experiments of general interest, including hematopoietic-specific knock-outs, tissue targeted dominant negatives, and possibly gene therapy vectors. Through the use of transgenic mice with chimeric GATA-1/lacZ reporter constructs, and DNase I hypersensitivity mapping experiments, the candidate and mentor have identified one such regulatory region that is required for high level expression of the GATA-1 gene in primitive and definitive erythrocytes and megakaryocytes. A number of experimental approaches to functionally analyze this region will be pursued. By a strategy of gene targeted deletion of this element, they have investigated the role of this sequence in its natural chromatin context. These mice die at the fetal liver stage of hematopoiesis and have abnormal colony formation when bone marrow or fetal liver cells are tested in in vitro colony formation assays. GATA-1 levels are decreased by not absent. They seek to establish the nature of the anemia in these mice and to explore the apparent gene dosage effect of GATA-1 in terminal erythroid maturation. Additional mice generated with the neomycin selection cassette removed will be analyzed in a similar manner to rule out possible confounding effects of the selectable marker on the phenotype. This system will allow systematic evaluation of other putative cis-elements as well.
