Functions of the SNAG Repression Domain in Oncogenesis

描述(申请人提供)：真核生物锌指蛋白的Snail-GFI1(SNAG)亚家族编码后生动物发育和内稳态途径的关键调节因子。脊椎动物的SNAG-ZF家族共有一个由5-7个Cys2-His2锌指组成的COOH末端DNA结合域和一个高度保守的NH2末端，该末端含有21个氨基酸的SNAG阻遏结构域。这个结构域是一个有效的、可转移的阻遏结构域。关于SNAG结构域介导的抑制机制，目前还一无所知。含有结构域的原癌基因GFI1(生长因子独立性-1)与T细胞胸腺瘤的发生有关。我们使用了GFI1的SNAG结构域，首先对SNAG介导的抑制进行了全面的突变分析，并使用这组突变在酵母双杂交筛选中区分了候选的SNAG-辅抑制子蛋白。我们发现了一种新的含有LIM结构域的蛋白AJUBA，它与野生型SNAG结构域结合，而不与缺乏抑制活性的突变体结合。SNAG-AJUBA相互作用在体内发生，并增强SNAG结构域介导的抑制作用。值得注意的是，AJUBA蛋白穿梭于细胞质和细胞核之间，可能代表着一种新的信号系统，它利用SNAG抑制结构域作为核受体。我们将通过以下具体目标进一步表征GFI1-AJUBA的相互作用：具体地说，我们将：1.鉴定和鉴定内源性GFI1-AJUBA复合体，重组、定位并确定体内和体外SNAG结构域-AJUBA相互作用的特异性。2.明确GFI1-AJUBA介导共抑制的机制、抑制位点的分子特征以及RAS信号对AJUBA共抑制的影响。3.利用依赖于SNAG-ZF和AJUBA功能的体内细胞增殖和分化系统，确定SNAG结构域与AJUBA相互作用的生物学相关性。4.纯化内源性GFI1和AJUBA复合体，确定其组成及其调控。

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.