Department of Plant Physiology, RWTH Aachen University, Aachen, Germany.
Nat Protoc. 2020 Mar;15(3):713-733. doi: 10.1038/s41596-019-0277-9. Epub 2020 Feb 10.
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.
真核基因转录与核小体的逐出和开放染色质的形成有关，这使得转录共激活因子和其他调节因子得以招募。因此，开放染色质是基因组中功能性调节 DNA 元件的标志。近年来，甲醛辅助分离调节元件（FAIRE）已被证明在鉴定各种真核生物（尤其是酵母、人类和小鼠）基因组中的开放染色质方面非常有效。然而，该方法在植物材料上的应用具有挑战性，并且少数可用的方案都有其缺点（例如，仅适用于特定的发育阶段）。在本方案扩展中，我们描述了一种可靠的 FAIRE 方案，用于成熟的拟南芥（Arabidopsis thaliana）叶片，该方案对原始方案进行了适应，以用于植物。与早期 FAIRE 方案相比，该方案扩展的主要区别在于染色质交联缓冲液中的甲醛浓度增加，应用重复真空以提高交联效率，以及 DNA 提取缓冲液的组成改变。该方案适用于未受胁迫和受胁迫植物的叶片染色质，并且可以在 1 周内完成。在这里，我们还描述了使用 qPCR 和下一代测序的下游分析。然而，该方案扩展也应该与 DNA 微阵列的下游杂交兼容。此外，只需进行一些微小的适应，就可以将该方案应用于其他拟南芥器官或植物物种。
