Komar Dorota N, Mouriz Alfonso, Jarillo José A, Piñeiro Manuel
Centro de Biotecnología y Genómica de Plantas (CBGP), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid.
Centro de Biotecnología y Genómica de Plantas (CBGP), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Universidad Politécnica de Madrid;
J Vis Exp. 2016 Jan 14(107):e53422. doi: 10.3791/53422.
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.
复杂的基因调控网络精心编排植物中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植物对环境信号和发育线索的响应。因此，深入了解控制植物基因表达的机制对于深刻理解植物中生物过程的调控方式至关重要。这里描述的染色质免疫沉淀（ChIP）技术是一种用于鉴定模式植物拟南芥基因或基因组区域中蛋白质DNA结合位点的方法。使用ChIP方案可以有效地分析与感兴趣的蛋白质（如转录因子、染色质蛋白或组蛋白的翻译后修饰形式）的DNA相互作用。该方法基于体内蛋白质-DNA相互作用的固定、染色质的随机片段化、用特异性抗体免疫沉淀蛋白质-DNA复合物以及通过PCR技术对与感兴趣蛋白质相关的DNA进行定量。在拟南芥中使用这种方法对于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。这种方法使得能够鉴定拟南芥染色质蛋白EBS与开花主基因FT调控区域的结合位点。通过ChIP分析还揭示了这种蛋白质对FT基因组区域中特定组蛋白标记积累的影响。
