Journal Article

A fast, efficient chromatin immunoprecipitation method for studying protein-DNA binding in Arabidopsis mesophyll protoplasts

Jeong Hwan Lee, Suhyun Jin, Wanhui Kim, Ji Hoon Ahn, Sun Young Kim

2017

Overview

Background Binding of transcription factors to their target sequences is a primary step in the regulation of gene expression and largely determines gene regulatory networks. Chromatin immunoprecipitation (ChIP) is an indispensable tool used to investigate the binding of DNA-binding proteins (e.g., transcription factors) to their target sequences in vivo. ChIP assays require specific antibodies that recognize endogenous target transcription factors; however, in most cases, such specific antibodies are unavailable. To overcome this problem, many ChIP assays use transgenic plants that express epitope-tagged transcription factors and immunoprecipitate the protein with a tag-specific antibody. However, generating transgenic plants that stably express epitope-tagged proteins is difficult and time-consuming. Results Here, we present a rapid, efficient ChIP protocol using transient expression in Arabidopsis mesophyll protoplasts that can be completed in 4 days. We provide optimized experimental conditions, including the amount of transfected DNA and the number of protoplasts. We also show that the efficiency of our ChIP protocol using protoplasts is comparable to that obtained using transgenic Arabidopsis plants. We propose that our ChIP method can be used to analyze in vivo interactions between tissue-specific transcription factors and their target sequences, to test the effect of genotype on the binding of a transcription factor within a protein complex to its target sequences, and to measure temperature-dependent binding of a transcription factor to its target sequence. Conclusions The rapid and simple nature of our ChIP assay using Arabidopsis mesophyll protoplasts facilitates the investigation of in vivo interactions between transcription factors and their target genes.

• Publisher: Springer Science and Business Media LLC,BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Antibodies / Arabidopsis / Arabidopsis; Chromatin immunoprecipitation; Protoplasts; Transcriptionfactor; Transient expression / Assaying / Binding / Biological Techniques / Biology (General) / Biomedical and Life Sciences / Chromatin / Chromatin immunoprecipitation / Deoxyribonucleic acid / DNA / DNA binding proteins / DNA-binding protein / Epitopes / Gene expression / gene expression regulation / gene regulatory networks / Genetic aspects / Genomes / genotype / Genotype & phenotype / Immunoglobulins / Immunoprecipitation / In vivo methods and tests / Kinases / Life Sciences / Localization / Mesophyll / Methodology / Methods / Nucleotide sequence / Plant culture / Plant Sciences / precipitin tests / Proteins / Protoplast / Protoplasts / QH301-705.5 / SB1-1110 / Signal transduction / Sodium / Studies / Temperature dependence / Temperature effects / transcription (genetics) / Transcription factor / Transcription factors / Transgenic plants / Transient expression