DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.
DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.
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DamID-seq：一种全基因组 DNA 甲基化方法，可捕获植物细胞中瞬时和稳定的 TF-DNA 相互作用。
DOI：
10.1007/978-1-0716-3354-0_7
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Coruzzi,GloriaM
中科院分区：
文献类型：
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作者：
Alvarez,JoséM;Hinckley,WillE;Leonelli,Lauriebeth;Brooks,MatthewD;Coruzzi,GloriaM
捕捉转录因子(Tf)与其全基因组靶标的动态和瞬时相互作用是一项重大的技术挑战。这是染色质免疫沉淀-测序(ChIP-SEQ)等生物化学方法普遍存在的问题，这些方法偏向于捕捉稳定的TF-靶基因相互作用。在这里，我们描述了如何使用DNA腺嘌呤甲基转移酶鉴定和测序(DAMID-seq)来捕获通过DNA甲基化而产生的瞬时和稳定的转铁蛋白靶标相互作用。DamID技术使用融合在DNA腺嘌呤甲基转移酶(DAM)上的TF蛋白。Coli.当在植物细胞中表达时，Dam-Tf融合蛋白将使Tf-DNA相互作用部位附近的腺嘌呤(A)碱基甲基化。通过这种方式，DamID会在Tf-靶基因启动子上产生一个永久、稳定的DNA甲基化标记，即使目标基因只被Dam-Tf融合蛋白短暂地“接触”。在这里，我们提供了一个循序渐进的方案，在分离的植物细胞中对任何感兴趣的Dam-Tf融合蛋白进行DamID-seq实验。我们还提供信息，使研究人员能够分析DamID-seq数据，以确定基因组中的TF结合位点。我们的方案包括Dam-Tf融合蛋白的载体克隆、植物细胞原生质体转染、DamID准备、文库准备和测序数据分析。本章概述的方案是在拟南芥中执行的，然而，本指南中开发的DAMID-SEQ工作流程广泛适用于其他植物和生物体。
Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants’ adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fused to a DNA adenine methyltransferase (Dam) fromE. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-target gene promoters, even if the target gene is only transiently “touched” by the Dam-TF fusion protein. Here we provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed inArabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.