Designer Transcription Activator Like Effector-Chromatin Affinity Purification (dTALE-ChAP) – an in planta Approach to Unravel the Protein Coverage at a Promoter of Choice
Final Report Abstract
The dTALE-ChAP method was to be developed in the project. Conceptually, this is a novel in vivo method that should allow the proteome of a desired promoter to be analyzed. In this method, GFP-tagged designer Transcription Activator Like Effectors (dTALEs) were used in transgenic Arabidopsis seedlings, which served as specific DNA-binding anchor proteins for Chromatin Affinity Purification (ChAP). The chromatin-associated proteins precipitated via the dTALEs were determined by mass spectrometry (MS). The aim was to identify proteins via dTALE-ChAP that bind to the promoter of the Flagellin 22 Induced Receptor Like Kinase 1 (pFRK1) gene or the Arabidopsis Response Regulator 5 (pARR5) gene. The expression of these genes is strongly increased by the respective ligand within 60 min (flg22 for FRK1; kinetin for ARR5). After metabolic 14N/15N labeling, proteins and transcription factors (TFs) that interact with pFRK1 or pARR5 in a ligand-dependent manner should also be quantified. Numerous preliminary experiments on e.g. expression, inducible nuclear localization, precipitability from nuclear extracts, in vivo binding of dTALEs to "their" specific DNA element showed that the dTALE-ChAP approach is possible in principle. Performing dTALE-ChAPs with specific dTALEs for pFRK1 and pARR5 in response to the presence of the ligand revealed proteins belonging to the GO domains "Heterochromatin organization", "Chromatin silencing" and "Negative regulation of gene expression". Regarding these proteins, there were no significant principle differences between pFRK1 and pARR5. TFs that would have been expected to bind to pFRK1 (e.g. WRKYs) and pARR5 (e.g. B-type ARRs) were not found. Our results suggest that (i) dTALE-ChAP can detect general protein changes in the organization of heterochromatin in promoters quantitatively, (ii) it does not seem to be sensitive enough for the detection of TFs at promoters and (iii) we were probably too late with our harvest time points after ligand application.
Publications
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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. Inaugural Dissertation, Universität Tübingen.
Stefan M. Fischer
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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. Inaugural Dissertation, Universität Tübingen.
Stefan M. Fischer
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The TALE- ChAP: a novel in planta method to identify proteins that associate with a promoter of choice. Molecular Biology of Plants-Konferenz (MBP2020).
Fischer S. M.; Schiele L.; Morbitzer R.; Lahaye T. & Brand LH, Harter K.
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The TALE-ChAP: a novel in planta method to identify proteins that associate with a promoter of choice. 5th RegioPlantScience Conference, University of Hohenheim.
Fischer S.M.; Schiele L.; Morbitzer R.; Lahaye T.; Brand L.H. & Harter K.
