COFACTORS AND FUNCTIONS OF HOMEODOMAIN REPRESSORS

描述(申请人提供)：基因转录的变化在癌症、大多数其他人类疾病的进展、衰老过程以及多细胞生物体的所有阶段的发育中都是重要的。详细了解这些变化是如何被调控的，是诊断工具和干预策略的基础。进一步的进步意味着新方法的前景，以及现有方法的有效性提高。重要的是，关于所涉及的基本过程仍有许多问题。序列特异的DNA结合蛋白及其招募到DNA的辅因子是最重要的转录调节因子。其中一个最大的蛋白质家族共享DNA结合的同源结构域基序。果蝇体内可用的工具使我们有可能在真实的活体环境中详细研究作用和相互作用的机制。这一建议是为了研究这个家族中几个成员的作用机制，以解决仍然存在的基本问题，即特定DNA靶点的识别是如何完成的，以及这种识别对辅助因子的招募以抑制转录、对介导调控的染色质变化以及对正在被调控的发育途径的影响。采用体内和体外相结合的方法，这些研究将提供更清楚的理解，了解蛋白质组合如何识别体内适当的靶点，以及这种识别对下游基因和途径调控的影响。该项目的具体目标是： 1)确定形成同源结构域蛋白的靶基因特异性的机制。研究从DNA结合到抑制直接靶基因草率配对的机制。 2)分析含有锌指DNA结合域的内相互作用蛋白的功能，并测试该蛋白在体内是否有助于靶基因的特异性或对特定靶点的激活/抑制功能。 3)鉴定和分析靶基因sloppy配对中跳过的含有同源结构域的抑制子的功能结合部位。确定在草率的发展过程中负责顺序抑制的机制，通过先偶跳过然后累加配对。

DESCRIPTION (provided by applicant): Changes in gene transcription are important in the progression of cancer, in most other human diseases, and in the aging process, as well as in the development of multicellular organisms at all stages. A detailed understanding of how such changes are regulated is the basis of both diagnostic tools and intervention strategies. Further advancement holds the promise of novel approaches, and of increased effectiveness of current approaches. Importantly, many questions remain about the fundamental processes involved. Sequence-specific DNA binding proteins and the cofactors that they recruit to the DNA are among the most important regulators of transcription. One of the largest such families of proteins share the DNA binding homeodomain motif. Tools available in Drosophila make it possible to study mechanisms of action and interaction in detail in a true in vivo context. This proposal is to study mechanisms of action of several members of this family, to address basic questions that remain about how recognition of specific DNA target sites is accomplished, and the consequences of that recognition for the recruitment of cofactors to repress transcription, for chromatin changes that mediate regulation, and for the developmental pathways that are being regulated. Using a combination of in vivo and in vitro approaches, these studies will provide a clearer understanding of how combinations of proteins recognize appropriate target sites in vivo, and the consequences of that recognition for the regulation of downstream genes and pathways. The Specific Aims of the project are: 1) To determine the mechanisms that generate target gene specificity for the homeodomain protein Engrailed. Investigate the mechanisms that lead from DNA binding to repression of the direct target gene sloppy paired. 2) To analyze the functions of an En-interacting protein that contains a zinc-finger DNA binding domain, and to test whether this partner contributes to target gene specificity, or to activation/repression function on specific target sites in vivo. 3) To identify and analyze functional binding sites for the homeodomain-containing represser Evenskipped in the target gene sloppy paired. Determine the mechanisms responsible for the sequential repression during development of sloppy paired by first Even-skipped and then Engrailed.