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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dc.contributor.advisor Harter, Klaus (Prof. Dr.)
dc.contributor.author Fischer, Stefan Markus
dc.date.accessioned 2018-11-13T10:01:52Z
dc.date.available 2018-11-13T10:01:52Z
dc.date.issued 2020-09-07
dc.identifier.other 172895066X de_DE
dc.identifier.uri http://hdl.handle.net/10900/84765
dc.identifier.uri http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-847650 de_DE
dc.identifier.uri http://dx.doi.org/10.15496/publikation-26155
dc.description.abstract 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. en
dc.language.iso en de_DE
dc.publisher Universität Tübingen de_DE
dc.rights ubt-podok de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=de de_DE
dc.rights.uri http://tobias-lib.uni-tuebingen.de/doku/lic_mit_pod.php?la=en en
dc.subject.classification Methode , Proteine , Gen de_DE
dc.subject.ddc 570 de_DE
dc.subject.other TALE en
dc.subject.other Chromatin Immuno Precipitation en
dc.subject.other Chromatin Affinity Purification en
dc.title 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 en
dc.type Dissertation de_DE
dcterms.dateAccepted 2018-10-25
utue.publikation.fachbereich Biologie de_DE
utue.publikation.fakultaet 7 Mathematisch-Naturwissenschaftliche Fakultät de_DE

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