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.

与特定启动子相关的蛋白质组及其翻译后调节的动力学对于响应发育和环境线索的活性至关重要。但是，植物中尚无技术可用的技术来实现特定启动子相关蛋白质组的体内测定。该项目旨在建立这种技术，即名为Designer转录激活剂，例如效应 - 染色质亲和力纯化（dtale-chap）。 dtale-chap基于GFP标记的dtales，在外部诱导的转移到核中后，识别选择启动子内的特定DNA区域，并用于保留附近的染色质。然后，通过质谱法（MS）分析了与染色质相关的蛋白质组及其翻译后修饰。dtale-chap的建立构成了四个步骤：（1）识别利益启动子的功能区域。 （2）设计和克隆几个特定的​​dtales，以覆盖整个启动子。 （3）检查dtales的细胞内定位及其在植物能力上与启动子中其目标部位结合的能力。 （4）代谢14n/15n标记拟南芥幼苗的定量dtale-chap以及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诱导的受体（PFRK1）基因的启动子的蛋白质组，因为已经有许多初步数据。随后，dtale-chap将扩展到拟南芥反应调节剂5（PARR5）基因的启动子。 ARR5启动子及其对跨性因素的调节是我们研究细胞分裂素信号传导的中心兴趣。从我们的研究中，我们期望对蛋白质组成的动力学及其在植物启动子的翻译后修饰以及对其功能含义的更深入了解。