Functions of the SNAG Repression Domain in Oncogenesis

描述（由申请人提供）：真核锌指（ZF）蛋白的Snail-GFI 1（SNAG）亚家族编码后生动物发育和稳态途径的关键调节因子。脊椎动物中的SNAG-ZF家族共有一个由5 - 7个Cys 2-His 2锌指组成的COOH末端DNA结合结构域和一个高度保守的含有21个氨基酸的SNAG抑制结构域的NH 2末端。该结构域是有效的、可转移的阻遏结构域。关于SNAG结构域介导的抑制机制还不清楚。原型含有SNAG结构域的癌基因，GFI 1（生长因子独立性-1）负责T细胞胸腺瘤的发展。我们使用GFI 1 SNAG结构域，首先对SNAG介导的阻遏进行了全面的诱变分析，并使用这组突变来区分酵母双杂交筛选中的候选SNAG辅阻遏蛋白。我们发现了一种新的，LIM结构域含有蛋白AJUBA结合野生型SNAG结构域，但不缺乏抑制活性的突变体。 SNAG-AjuBA相互作用在体内发生并增强SNAG结构域介导的抑制。 值得注意的是，AjuBA蛋白穿梭于细胞质和细胞核之间，并可能代表一种新的信号系统，该系统利用SNAG抑制结构域作为核受体。我们将通过执行以下具体目标来进一步表征GFI 1-AJUBA相互作用：具体而言，我们将：1.鉴定和表征内源性GFI 1-AJUBA复合物，在体外和体内重建、绘制和确定SNAG结构域-AJUBA相互作用的特异性。2.阐明GFI 1-AJUBA介导的阻遏机制、阻遏位点的分子特征以及Ras信号对AJUBA共阻遏的影响。3.使用依赖于SNAG-ZF和AJUBA功能的体内细胞增殖和分化系统来定义SNAG结构域-AJUBA相互作用的生物学相关性。4.纯化内源性GFI 1和AjuBA复合物，并确定其组分及其调控。

DESCRIPTION (provided by applicant): 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.