Abstract
Eukaryotic gene transcription is associated with the eviction of nucleosomes and the formation of open chromatin, which enables the recruitment of transcriptional coactivators and other regulatory factors. Open chromatin is thus a hallmark of functional regulatory DNA elements in genomes. In recent years, formaldehyde-assisted isolation of regulatory elements (FAIRE) has proven powerful in identifying open chromatin in the genome of various eukaryotes, particularly yeast, human, and mouse. However, it has proven challenging to adapt the FAIRE protocol for use on plant material, and the few available protocols all have their drawbacks (e.g., applicability only to specific developmental stages). In this Protocol Extension, we describe a reliable FAIRE protocol for mature Arabidopsis (Arabidopsis thaliana) leaves that adapts the original protocol for use on plants. The main differences between this protocol extension and the earlier FAIRE protocol are an increased formaldehyde concentration in the chromatin crosslinking buffer, application of a repeated vacuum to increase crosslinking efficiency, and altered composition of the DNA extraction buffer. The protocol is applicable to leaf chromatin of unstressed and stressed plants and can be completed within 1 week. Here, we also describe downstream analysis using qPCR and next-generation sequencing. However, this Protocol Extension should also be compatible with downstream hybridization to a DNA microarray. In addition, it is likely that only minor adaptations will be necessary to apply this protocol to other Arabidopsis organs or plant species.
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Data availability
The unprocessed data of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank S.F. Beyer for help with the figures. U.C. was supported by a grant from the German Research Foundation (CO 186/13-1) and by the Excellence Initiative of the German federal and state governments.
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U.C. initiated and supervised the project. M.R.J. introduced S.B. to chromatin techniques. S.B. and E.-M.R.-M. performed the experiments. U.C. wrote the article with contributions from the other authors.
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Peer review information Nature Protocols thanks Jeremy M. Simon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Related links
Key reference(s) using this protocol
Baum, S. et al. Plant Physiol. 181, 817–833 (2019): https://doi.org/10.1104/pp.19.00673
Schillheim, B. et al. Plant Physiol. 176, 2395–2405 (2018): https://doi.org/10.1104/pp.17.00124
This protocol is an extension to: Nat. Protoc. 7, 256–267 (2012): https://doi.org/10.1038/nprot.2011.444
Integrated supplementary information
Supplementary Figure 1 Open chromatin in the 5′ regulatory region and around the TSS of the PR1 gene in systemic leaves relative to the UBIQUITIN5 promoter.
The experiment was done three times (n = 3). ***, P = 0.001; **, P = 0.01. Measurements were taken from distinct samples. Normal distribution was assumed for all statistical analyses. Unpaired Student´s t tests (two-sided) was applied using SigmaStat 4.0 (Systat Software, Inc.) to determine whether the observed differences were statistically significant. Changes were considered statistically significant when P ≤ 0.01. Psm, Pseudomonas syringae pv. maculicola; TSS, transcription start site; bp, base pairs; R1 - R3, biological replicates 1–3.
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Supplementary Fig. 1 and Supplementary Table 1.
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Baum, S., Reimer-Michalski, EM., Jaskiewicz, M.R. et al. Formaldehyde-assisted isolation of regulatory DNA elements from Arabidopsis leaves. Nat Protoc 15, 713–733 (2020). https://doi.org/10.1038/s41596-019-0277-9
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DOI: https://doi.org/10.1038/s41596-019-0277-9
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