MECHANISMS OF TRANSCRIPTIONAL REGULATION

转录调控机制

• 批准号: 2392021
• 负责人: James L. Manley
• 金额: $ 23.86万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392021
• 关键词: Drosophilidae; chimeric proteins; developmental genetics; gene induction /repression; gene mutation; homeobox genes; intermolecular interaction; invertebrate embryology; larva; molecular chaperones; phosphoproteins; protein structure function; regulatory gene; site directed mutagenesis; tissue /cell culture; transcription factor; transfection

DESCRIPTION: The overall goal of this research project is to understand some of the detailed molecular mechanisms involved in regulation of transcription in higher eukaryotic cells. Attention will focus on several Drosophila proteins that were initially identified by genetic methods as being important in controlling pattern formation in early embryogenesis, and which are now known to be transcriptional regulators. For the purposes of this project, these proteins will be studied as prototypical regulators that illustrate general mechanisms of gene control. That is, the aim is not to understand detailed pathways of developmental regulation in the Drosophila embryo, but rather to understand underlying biochemical mechanisms. Specifically, ongoing studies dealing with three specific regulators, or types of regulators, will be pursued. All the experiments will involve transfection assays in cultured cells, protein-protein interaction studies and in vitro transcription experiments with purified components. 1) Mechanisms of transcriptional activation will be studied using several previously described homeodomain activators. Recent experiments have focused on understanding how the glutamine-rich activation region from the Fushi tarazu protein functions (in the context of a GAL4-ftz fusion), and have provided evidence that a novel, direct interaction with the general transcription factor TFIIB is essential. Experiments to investigate further the mechanism by which this very strong, non-acidic activator functions will be performed. Using the GAL4-ftz activator together with others (e.g., GAL4-VP16), possible mechanisms of transcriptional synergy will also be examined. 2) Mechanisms of transcriptional repression will be investigated, using the homeodomain proteins Even-skipped (Eve) and Engraved (En). Both proteins are among the best characterized transcriptional repressors, and available data suggest both, especially Eve, function as direct, or active, repressors that interfere with or prevent assembly of the preinitiation complex. Recent experiments indicating that Eve repression which involves a direct, functional interaction with the TATA binding protein, TBP, will be extended to understand the details of this interaction, as well as how it leads to transcriptional repression. A possible role of phosphorylation in modulating activity will also be investigated. 3) Mechanisms of transcription factor regulation will be investigated by continuing studies on Dorsal (D1), a rel/NF-kappaB family member. Previous work has indicated that regulated nuclear transport of D1 can be reproduced in transfected Schneider cells, and that the activities of at least two genetically defined regulators of D1, Toll and tube, can be detected and analyzed. Those studies led to a model involving activation of D1 by direct phosphorylation, which both induces nuclear transport and enhances the transcriptional activating potential of D1, with recent studies indicating that the tube protein functions as both a chaperon and coactivator of D1. Experiments to examine further the mechanisms by which D1 nuclear transport is regulated, and its activity modulated, will be performed.

描述：该研究项目的总体目标是了解 一些涉及调节的详细分子机制 在较高的真核细胞中转录。注意将集中在 几种最初通过遗传鉴定的果蝇蛋白 方法对于早期控制模式形成很重要 胚胎发生，现在已知是转录调节剂。 出于该项目的目的，这些蛋白质将被研究为 典型调节剂说明基因的一般机制 控制。也就是说，目的不是了解 果蝇胚胎中的发展调节，而是 了解潜在的生化机制。具体来说，正在进行 处理三个特定监管机构或调节器类型的研究， 将被追捕。所有实验将涉及转染测定法 在培养的细胞中，蛋白质 - 蛋白质相互作用研究和体外 具有纯化成分的转录实验。 1）机制 先前将研究转录激活 描述了同源域激活剂。最近的实验重点是 了解如何来自谷氨酰胺富含谷氨酰胺的激活区域 塔祖蛋白功能（在GAL4-FTZ融合的背景下），并具有 提供了与一般直接相互作用的新颖的证据 转录因子TFIIB至关重要。 进行研究的实验 此外，这种非常强，非酸性激活剂的机制 将执行功能。将gal4-ftz激活剂与 其他（例如GAL4-VP16），转录协同作用的可能机制 也将被检查。 2）转录抑制的机制将 使用同源域蛋白（EVE）和 雕刻（en）。两种蛋白质都是最好的特征 转录阻遏物和可用数据都建议，尤其是 前夕，充当直接或活动的阻遏物，会干扰或 防止组装前启动络合物。最近的实验 表明涉及直接功能性的夏娃抑制 与TATA结合蛋白TBP的相互作用将扩展到 了解这种互动的细节，以及如何导致 转录抑制。磷酸化在 调节活动也将进行研究。 3）机制 转录因子调节将通过继续研究 Rel/NF-kappab家庭成员的背侧研究（D1）。以前的工作有 表明可以在 转染的施耐德细胞，至少两个活性 可以检测到D1，Toll和Tube的遗传定义调节剂，并 分析。这些研究导致了涉及D1激活D1的模型 直接磷酸化，既诱导核转运和 增强了D1的转录激活潜力，最近 研究表明该管蛋白既是伴侣 和D1的共激活因子。实验以进一步研究机制 哪种D1核转运受到调节，其活性调节， 将执行。