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

蛋白质组的动力学及其翻译后调控与特定的启动子相关，对于其响应发育和环境线索的活性至关重要。然而，目前还没有技术可以在植物体内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，命名为转录激活因子样效应-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后，识别选定启动子内的特定DNA区域，并用于沉淀附近的染色质。然后，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)确定感兴趣的启动子的功能区域。(2)设计和克隆几个特定的dtale，覆盖整个启动子。(3)检查dTALEs的细胞内定位及其在植物中与启动子中目标位点结合的能力。(4)对拟南芥幼苗进行代谢14N/ 15n后的dTALE-ChAP定量标记，分析亲和纯化的蛋白质组及其修饰。首先，将分析鞭毛蛋白22诱导受体样激酶（pFRK1）基因启动子处的蛋白质组，因为已经有了大量的初步数据。随后，dTALE-ChAP将扩展到拟南芥应答调节因子5 （pARR5）基因的启动子。ARR5启动子及其通过反式作用因子的调控是我们研究细胞分裂素信号传导的核心兴趣。从我们的研究中，我们期望对植物启动子中蛋白质组成及其翻译后修饰的动力学机制有更深入的了解，并对其功能意义有更深入的了解。

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.