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Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System   

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Original research article

A brief version of this protocol appeared in:
The Plant Cell
Apr 2017

Abstract

Chromatin Affinity Purification (ChAP) is widely used to study chromatin architecture and protein complexes interacting with DNA. Here we present an efficient method for ChAP from Arabidopsis thaliana rosette leaves, in which in vivo biotinylation system is used. The chromatin is digested by Micrococcal Nuclease (MNase), hence the distribution of nucleosomes is also achieved. The in vivo biotinylation system was initially developed for Drosophila melanogaster (Mito et al., 2005), but the presented protocol has been developed specifically for Arabidopsis thaliana (Sura et al., 2017).

Keywords: Chromatin affinity purification, ChAP-qPCR, Chromatin immunoprecipitation, ChIP, Chromatin MNase digestion, Nucleosome occupancy, Histone distribution, Arabidopsis thaliana

Background

Chromatin Immunoprecipitation (ChIP) became one of the most important and commonly used technique to study chromatin structure and organization. However, it requires good-quality antibodies, which will not cross-react with non-specific targets. This is relatively difficult to achieve in plants, which contain cell wall and are rich in photosynthesis-related compounds and proteins frequently causing cross-reactivity problems. On the other hand, obtaining stable transgenic organisms is a routine and easy strategy in plants. For these reasons most plant researchers choose gene tagging, where fusion proteins are obtained and used to study chromatin in an approach alternative to ChIP, that is Chromatin Affinity Purification (ChAP). The ChAP technique has proven to be extremely effective in plant chromatin studies (Zentner and Henikoff, 2014). Moreover, it is usually cheaper than classical ChIP as it does not require generation of antibodies, and is often more effective than ChIP as tags are recognized with higher affinity than antibodies raised directly against proteins of interests. One disadvantage of ChAP is that it cannot be used to study post-translational histone modifications. In the presented protocol, proteins are tagged with a short, Biotin ligase recognition peptide (BLRP), which is in vivo biotinylated by Escherichia coli BirA biotin ligase (de Boer et al., 2003). Consequently, the tagged protein is purified using streptavidin-based purification systems (e.g., Dynabeads M-280 Streptavidin, Invitrogen). As streptavidin has an extraordinarily high affinity for biotin (dissociation constant on the order of 10-14 mol/L), the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature (Green, 1975). Alternatively, the presented protocol can be successfully applied for ChAP of proteins labeled with other tags (e.g., MYC, GFP, HA, FLAG) if a suitable system for final purification is used.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Sura, W. and Ziolkowski, P. A. (2018). Chromatin Affinity Purification (ChAP) from Arabidopsis thaliana Rosette Leaves Using in vivo Biotinylation System. Bio-protocol 8(1): e2677. DOI: 10.21769/BioProtoc.2677.
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