The goal of this proposal is to comprehensively characterize the role of the transcription factor, GA-Binding Protein (GABP). in myeloid differentiation and gene expression. Transcription factors regulate development of myeloid cells (granulocytes and monocytes) from hematopoietic progenitor cells, and defects in transcription factors are associated with acute myelogenous leukemia. GABP is 1 of a limited number of transcription factors that regulate myeloid genes. It is a tetrameric complex that consists of 2 distinct proteins: 1) GABPalpha, an ets factor which binds to DNA, and 2) GABPbeta, a Notch-related protein which contains transcription activation and multimerization domains. GABP transcriptionally activates CD 18 (beta2 leukocyte integrin) and other myeloid genes; it is required for transcriptional activation of CD18 in response to retinoic acid. As granulocytes and monocytes differentiate, distinct isoforms of GABPbeta with specific transcriptional activation properties are expressed; conditional expression some GABP beta isoforms induces granulocytic differentiation and characteristic gene expression. The proposal will test the hypothesis that GABP mediates an alternative pathway to granulocytic differentiation and that specific GABPbeta isoforms are required during myeloid differentiation. GABP cooperates with PU.1, Sp1, RARalpha, and p300 to activate CD18. This proposal will use pull-down assays, co-immunoprecipitation, and chromatin immunoprecipitation to define the physical and functional interactions of GABP with other key myeloid transcription factors and co-activators. It will test the hypothesis that GABP participates in an enhanceosome - a multiprotein complex that regulates myeloid gene expression. Mice were generated with loxP recombination sites that flank the GABPalpha ets domain. GABPalpha is efficiently deleted in vivo and in vitro; homozygous deletion of GABPalpha in mice causes an early embryonic lethal defect. Selective disruption of GABPalpha in myeloid cells indicates that they require GABP for survival and/or differentiation. Downstream targets of GABP that may account for cellular defects of GABPalpha null cells have been identified. The proposal will test the hypothesis that GABP is required for granulocytic survival and/or differentiation and it will define mechanisms by which GABP drives myeloid cell development. These studies will provide a comprehensive demonstration of the role of GABP in myeloid gene expression and provide important new information regarding transcription factor and co-activator interactions that regulate myeloid differentiation.
