Designer Transcription Activator Like Effector-Chromatin Affinity Purification (dTALE-ChAP) – an in planta Approach to Unravel the Protein Coverage at a Promoter of Choice

设计转录激活剂样效应子染色质亲和纯化 (dTALE-ChAP) – 一种揭示所选启动子处蛋白质覆盖的植物内方法

• 批准号: 426152216
• 负责人: Professor Dr. Klaus Harter
• 金额: --
• 依托单位: Abteilung für Pflanzenphysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426152216?language=en
• 关键词: Designer; Transcription; Activator; Like; Effector

The dynamics of the proteome and its post-translational regulation, which is associated with a specific promoter, is crucial for its activity in response to developmental and environmental cues. However, there is no technique available in plants yet that enables the quantitative in vivo determination of specific promoter-associated proteomes. The project aims to establish such a technique, named designer Transcription Activator Like Effector-Chromatin Affinity Purification (dTALE-ChAP). dTALE-ChAP is based on GFP-tagged dTALEs that, after externally induced transfer into the nucleus, recognize specific DNA regions within a promoter of choice and are used to precipitate the nearby chromatin. Then, the chromatin-associated proteome and its post-translational modification are analyzed by mass spectrometry (MS).The establishment of dTALE-ChAP comprises four steps: (1) Identification of the functional area of the promoter of interest. (2) Design and cloning of several specific dTALEs to cover the entire promoter. (3) Examination of the dTALEs´ intracellular localization and their in planta capacity to bind to their target site in the promoter. (4) Implementation of the quantitative dTALE-ChAP after metabolic 14N/15N-labelling of Arabidopsis seedlings and analysis of the affinity-purified proteome and its modification. At first, the proteome at the promoter of the Flagellin 22 Induced Receptor Like Kinase (pFRK1) gene will be analyzed, as numerous preliminary data are already available. Subsequently, dTALE-ChAP will be extended to the promoter of the Arabidopsis Response Regulator 5 (pARR5) gene. The ARR5 promoter and its regulation by trans-acting factors are of central interest in our investigation of cytokinin signaling. From our studies, we expect extensive mechanistic insights into the dynamics of protein composition and its post-translational modifications at plant promoters and deeper knowledge about its functional implications.

蛋白质组的动态及其翻译后调节，这是与一个特定的启动子，是至关重要的，其活动在响应发育和环境的线索。然而，目前还没有一种技术可以在植物中定量测定特定的启动子相关蛋白质组。该项目旨在建立这样一种技术，命名为设计师转录激活因子样受体-染色质亲和纯化（dTALE-ChAP）。dTALE-ChAP是基于GFP标记的dTALE，在外部诱导转移到细胞核中后，识别所选启动子内的特定DNA区域，并用于沉淀附近的染色质。然后，利用质谱分析染色质相关蛋白质组及其翻译后修饰。dTALE-ChAP的建立包括四个步骤：（1）鉴定目的启动子的功能区。(2)设计和克隆几个特定的dTALE以覆盖整个启动子。(3)检查dTALE的细胞内定位及其在植物中与启动子中的靶位点结合的能力。(4)拟南芥幼苗代谢14 N/15 N标记后定量dTALE-ChAP的实施和亲和纯化蛋白质组及其修饰的分析。首先，将分析鞭毛蛋白22诱导受体样激酶（pFRK 1）基因启动子处的蛋白质组，因为已经有许多初步数据。随后，dTALE-ChAP将延伸至拟南芥应答调节子5（pARR 5）基因的启动子。ARR 5启动子及其反式作用因子的调控是我们研究细胞分裂素信号的核心。从我们的研究中，我们期望广泛的机制洞察蛋白质组成的动态及其在植物启动子的翻译后修饰和更深入的了解其功能的影响。