COFACTORS AND FUNCTIONS OF HOMEODOMAIN REPRESSORS

描述（由申请人提供）： 基因转录的变化在癌症的进展中是重要的，在大多数其他人类疾病中，在衰老过程中，以及在多细胞生物体发育的各个阶段。详细了解这些变化是如何调节的是诊断工具和干预战略的基础。进一步的进步有望产生新的方法，并提高当前方法的有效性。重要的是，关于所涉及的基本过程仍然存在许多问题。序列特异性DNA结合蛋白和它们向DNA募集的辅因子是转录的最重要调节因子之一。最大的此类蛋白质家族之一共享DNA结合同源结构域基序。在果蝇中可用的工具使得在真实的体内环境中详细研究作用和相互作用的机制成为可能。这个提议是研究这个家族的几个成员的作用机制，以解决仍然存在的关于如何识别特定DNA靶位点的基本问题，以及这种识别对辅助因子的招募以抑制转录、对介导调节的染色质变化以及对正在被调节的发育途径的影响。使用体内和体外方法的组合，这些研究将提供一个更清晰的了解蛋白质的组合如何识别适当的靶位点在体内，以及下游基因和途径的调控识别的后果。该项目的具体目标是： 1)确定同源结构域蛋白Engrailed产生靶基因特异性的机制。研究从DNA结合到直接靶基因草率配对的抑制的机制。 2)分析含有锌指DNA结合结构域的En相互作用蛋白的功能，并测试该配偶体是否有助于靶基因特异性或体内特定靶位点的激活/抑制功能。 3)目的：鉴定和分析靶基因中含有同源结构域的阻遏蛋白Evenskipped的功能结合位点。确定的机制，负责顺序抑制在发展过程中的草率配对的第一个偶数跳过，然后Engrailed。

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.