Several zinc finger genes are known to function in hematopoiesis. They may play important roles for cell fate determination in the hematopoietic system and they also may have the potential to play roles in neoplastic transformation. The current application is concerned with elucidation of the mechanism by which the myeloid zinc finger gene MZF-1 regulates normal hematopoiesis and how modification of its expression may affect leukemogenesis. In the first aim a biochemical dissection of the transcriptional activator and repressor domains of MZF-1 are proposed. By using deletion, truncation and substitution mutagenesis a structure function analysis of the activation domain, the Gly-Pro rich region and the zinc finger domains will be performed. GAL4 assays and heterologous promoter assays will be used to assess the properties of the mutants. Both the transcription activation and repression properties of the MZF-1 mutants as well as the oncogenic potential of the mutants will be determined in appropriate cell systems. In the second specific aim, first, proteins which interact with MZF-1 during transcriptional activation and repression will be identified by using the two hybrid system. Second, genes up or down regulated in NIH3T3 and FDCP1 cells transformed by MZF-1 will be identified by the differential display methodology. In the third specific aim, a role for MZF-1 in leukemogenesis will be investigated. For this purpose first, transgenic mice in which the MZF-1 gene is expressed driven by several promoters specifically active in hematopoietic cells will be constructed and the effect of the transgene on hematopoiesis will be investigated by standard approaches. Alternatively, the MZF-1 gene will be expressed in hematopoietic cells upon retroviral gene transfer into bone marrow cells in vitro using in the PLXSN vector developed by Dusty Millers lab. Subsequently, these infected progenitor cell populations will be transplanted into lethally irradiated congenic recipient mice and animals will be observed for the formation of hematopoietic neoplasm.
