Much of hematopoietic development is controlled by transcriptional regulation of those genes encoding for the phenotypic changes of blood cell maturation. Transcription itself is governed by various transcription factors. A myeloid-specific gene, MZF-l, was cloned which belongs to the zinc finger family of transcription factors. MZF-l was found to be important in granulopoiesis. The purpose of this proposal is to test the hypothesis that MZF-l regulates a portion of the genes activated during granulopoiesis. The first part of the study will entail isolation of the DNA-binding site of MZF-l. This will be done using mobility shift selection and PCR amplification of degenerate oligonucleotides. Known genes having this sequence in their promoter/enhancer regions will be identified by 'database searching. This sequence will be tested for its ability to stimulate transcription by cotransfection of an MZF-l expression vector with a reporter gene containing this sequence. The second part of this study will be to define the structural determinants of MZF-l binding to the specific DNA sequence. This will be analyzed using oligonucleotide- directed mutagenesis of the MZF-l protein. In the third part of this study stable transfectants of non-myeloid cells will be isolated containing an expression vector forcing over-production of sense MZF-l mRNA. These transfectants will be analyzed for myeloid phenotypic changes using flow cytometric analysis of myeloid surface antigens and northern analysis of myeloid-specific genes. In the final part of this investigation stable transfectants will be constructed of myeloid cell lines containing vectors forcing the over-expression of anti-sense MZF-l. Changes in phenotype produced by this abrogation of MZF-l activity will again be assessed using flow cytometry and northern analysis. Thus, this investigation proposes to-identify genes activated by MZF-l during granulopoiesis.
