Designer Transcription Activator Like Effector - Chromatin Affinity Purification (dTALE-ChAP) a novel in planta method to unravel the protein coverage at a promoter of choice

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Dokumentart: Dissertation
Date: 2020-09-07
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biologie
Advisor: Harter, Klaus (Prof. Dr.)
Day of Oral Examination: 2018-10-25
DDC Classifikation: 570 - Life sciences; biology
Keywords: Methode , Proteine , Gen
Other Keywords:
Chromatin Immuno Precipitation
Chromatin Affinity Purification
License: Publishing license including print on demand
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he novel in vivo method developed in this work, allows to analyze the proteome associated with any promoter of interest and is called dTALE-ChAP. This method makes use of a set of designer Transcription Activator Like Effectors (dTALEs), designed as bait proteins for Chromatin Affinity Purification (ChAP) with subsequent mass spectrometry (MS). To demonstrate the use of the dTALE-ChAP, stable transformed dTALE-expressing Arabidopsis thaliana lines were used. The target of choice to establish the method was the well-known promoter of the Flagellin22 induced Receptor Like Kinase 1 (pFRK1). To establish the method, several pretests had to be performed. First, expression of the dTALEs and their dexamethasone (DEX)-inducible nuclear translocation was confirmed in transgenic Arabidopsis thaliana lines by microscopy. Second, it was demonstrated by promoter-reporter gene assays in Arabidopsis protoplasts, that dTALEs specifically bind to their DNA target sequence, derived from the pFRK1. Third, it was shown by Chromatin Immuno-Precipitation, that a dTALE can precipitate pFRK1 fragments from nuclear extracts of transgenic Arabidopsis lines. Finally, the dTALE-ChAP was performed and several proteins including histones were identified to be associated with pFRK1. Thus, the dTALE-ChAP was successfully established and such a method was used for the first time in plants. This new method allows to analyze the dynamics and post-translational modifications of DNA associated proteins over time in any organism. In future, methods like the dTALE-ChAP will help to better understand transcriptional regulation.

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