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

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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.