The Snail-GFI1 (SNAG) subfamily of eukaryotic zinc finger (ZF) proteins encodes key regulators of developmental and homeostatic pathways in metazoans. The SNAG-ZF family in vertebrates share a COOH-terminal DNA binding domain composed of 5 -7 Cys2-His2 zinc fingers and a highly conserved NH2- terminus which contains the 21 amino acid SNAG repression domain. This domain is a potent, transferable repression domain. Nothing is known about the mechanisms of SNAG domain-mediated repression. The prototype SNAG domain-containing oncogene, GFI1 (growth-factor independence-1) is responsible for development of T-cell thymomas. We used the GFI1 SNAG domain and first performed a comprehensive mutagenic analysis of SNAG-mediated repression and used this set of mutations to distinguish candidate SNAG-corepressor proteins in a yeast two hybrid screen. We discovered a novel, LIM domain containing protein AJUBA which binds to wild-type SNAG domain but not to mutants which lack repression activity. The SNAG-AJUBA interaction occurs in vivo and enhances SNAG domain mediated repression. Remarkably, the AJUBA protein shuttles between the cytoplasm and the nucleus and may represent a novel signaling system which utilizes the SNAG repression domain as the nuclear receptor. We will further characterize the GFI1-AJUBA interaction by performing the following specific aims: Specifically we will: 1. Identify and characterize an endogenous GFI1-AJUBA complex, reconstitute, map and determine the specificity of the SNAG domain-AJUBA interaction in vitro and in vivo. 2. Define the mechanism of GFI1-AJUBA mediated repression, the molecular characteristics of the repressed locus and the influence of Ras signaling on AJUBA co-repression. 3. Define the biological relevance of SNAG domain-AJUBA interactions using in vivo cell proliferation and differentiation systems which are dependent upon SNAG-ZFs and AJUBA function. 4. Purify an endogenous GFI1 and AJUBA complex and define the components and their regulation. ? ?
