SCL GENE AND HEMATOPOIETIC DEVELOPMENT

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The proposed research will investigate the expression and functions of the protein products of the SCL gene during hematopoietic development in the mouse. This gene was first identified at the breakpoint of a chromosomal translocation in human T-cell leukemia and is a member of a family of transcription factors whose members are recognized to be regulators of differentiation. The results of these experiments should provide insight into basic mechanisms of hematopoiesis. Since activation of this gene by chromosomal rearrangement is the most common genetic lesion in acute T-cell leukemia, these studies should also contribute to our understanding of leukemogenesis. The first specific aim of this work is to characterize the translational products of the murine SCL gene in hematopoietic cells. The protein products of the murine SCL gene will be characterized in hematopoietic cell lines and mouse tissues by radioimmunoprecipitation and Western blot analysis. Subcellular localization of SCL proteins will be determined by transient expression in COS cells and in cell lines stably expressing individual proteins. The second specific aim is to characterize the transcriptional and translational products of the murine SCL gene during hematopoietic development. SCL mRNA expression will be studied with RNase protection, in situ hybridization, and the polymerase chain reaction (PCR) and SCL protein expression by immunocytochemistry in staged mouse embryos. The transcriptional and translational products of the gene will also be characterized in an in vitro model of hematopoietic development using embryonic stem (ES) cell-derived embryoid bodies. The third specific aim is to define the actions of SCL proteins in hematopoietic development. This will be investigated in gain of function and loss of function mutants of ES cells made to undergo differentiation. The fourth specific aim is to determine the ability of SCL proteins to function as transcriptional regulators during hematopoietic development. The ability of SCL proteins to transactivate artificial and, if identified, physiologic targets will be determined. The possibility that certain SCL proteins may have different transcriptional potencies and act as transcriptional antagonists will be investigated. Cellular targets will be identified using a functional strategy based on the isolation of SCL-inducible promoters, and the ability of different SCL isoforms to activate such promoters will be defined.