Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo
Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo
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DOI：
10.3791/53422
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发表时间：
2016-01-01
影响因子：
1.2
通讯作者：
Pineiro, Manuel
中科院分区：
文献类型：
--
作者：
Komar, Dorota N.;Mouriz, Alfonso;Pineiro, Manuel
复杂的基因调控网络协调了植物的生物过程和发育转变。基因的选择性转录激活和沉默介导了植物对环境信号和发育线索的反应。因此，深入了解控制植物基因表达的机制对于深入了解植物的生物过程是如何被调节的至关重要。本文描述的染色质免疫沉淀（ChIP）技术是鉴定模式物种拟南芥基因或基因组区域中蛋白质的dna结合位点的一种方法。通过ChIP协议，可以有效地分析感兴趣的蛋白质（如转录因子、染色质蛋白或翻译后修饰的组蛋白）与DNA的相互作用。该方法基于体内蛋白质-DNA相互作用的固定，染色质的随机碎片化，蛋白质-DNA复合物与特异性抗体的免疫沉淀，以及通过PCR技术定量与感兴趣的蛋白质相关的DNA。这种方法在拟南芥中的应用为揭示控制多种植物生物过程的转录调控机制做出了重大贡献。这种方法可以鉴定出拟南芥染色质蛋白EBS与开花FT主基因调控区域的结合位点。通过ChIP分析，还揭示了该蛋白在FT基因组区域特定组蛋白标记积累中的影响。
Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.