GTPases of the Rac family are important regulators of cytoskeletal remodeling in response to signals received via growth factor responses. The expression patterns of these GTPases are tightly regulated in response to cytochalasin B exposure. In contrast, Rac2 expression is restricted to hematopoietic cells, and is further induced upon terminal myeloid cell differentiation and T cell stimulation. Furthermore, there appears to be cross-talk between the expression of the Rac1 and Rac2 genes, as Rac1 expression is dysregulated in Rac2-null cells.
The specific aims of this proposal are:
(AIM #1) Identify genetic elements and cognate transcription factors responsible for restriction of Rac2 expression to hematopoietic cells. Cytosine methylation patterns and DNase hypersensitive sites surrounding the Rac2 locus will be determined. Bacterial artificial chromosome clones carrying the human Rac2 gene locus will be introduced into mice and murine cell lines, and transgene expression assessed by RNase protection assays in order to identify cis-elements necessary and sufficient to direct lineage-specific expression. In vitro DNA-binding protein assays will be performed to detect and identify transcription factors that interact with critical cis- elements, and novel factors will be molecularly cloned by either ligand screening or biochemical purification approaches.
(AIM #2) Analyze cross-talk between the Rac1 and Rac2 genes. Biochemical studies will be conducted to determine the level at which expression of the Rac1 gene is modulated in response to Rac2 levels. The Rac1 promoter will be cloned and functionally characterized, and critical cis-elements and cognate trans-factors will be identified. The molecular basis for dysregulated Rac1 expression in response to Rac 2 levels will be determined by introducing Rac1 promoter/luciferase reporter genes into primary murine blood cell cultures derived from Rac2-null mice.
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