Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)
Identification of Proteins Interacting with Genomic Regions of Interest in vivo Using Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (enChIP)
复制标题
使用工程 DNA 结合分子介导的染色质免疫沉淀 (enChIP) 鉴定体内与感兴趣的基因组区域相互作用的蛋白质
DOI：
10.21769/bioprotoc.1124
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
H. Fujii
中科院分区：
文献类型：
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作者：
T. Fujita;H. Fujii
[摘要]阐明基因组功能的分子机制需要鉴定与体内感兴趣的基因组区域相互作用的分子。为此，分离保留分子相互作用的靶区域是有用的。我们建立了基因座特异性染色质免疫沉淀（ChIP）技术，其由插入ChIP（iChIP）和工程化DNA结合分子介导的ChIP（enChIP）组成，用于分离靶基因组区域（Hoshino和Fujii，2009; Fujita和Fujii，2011; Fujita和Fujii，2012; Fujita和Fujii，2013 a; Fujita和Fujii，2013 b; Fujita等人，2013年）。与分离的基因组区域相互作用的分子的鉴定和表征有助于理解靶基因组区域功能的分子机制。在这里，我们描述了enChIP，其中工程化的DNA结合分子，如锌指蛋白、转录激活因子样（TAL）蛋白和催化失活的Cas9（dCas 9）加小向导RNA（gRNA），用于靶基因组区域的亲和纯化。enChIP的方案如下：1.产生锌指蛋白、TAL或dCas 9加gRNA以识别感兴趣的基因组区域中的DNA序列。2.工程化的DNA结合分子与标签和核定位信号（NLS）融合，并在待分析的细胞中表达。3.如果需要，将所得细胞交联并裂解，并将DNA片段化。4.对包含工程化DNA结合分子的复合物进行亲和纯化，例如免疫沉淀。分离的复合物保留与感兴趣的基因组区域相互作用的分子。5. DNA、RNA或蛋白质的反向交联和随后的纯化允许这些分子的鉴定和表征。在该方案中，我们描述了具有TAL蛋白的enChIP以分离感兴趣的基因组区域并通过质谱分析相互作用的蛋白（Fujita et al.，2013年）。
[Abstract] Elucidation of molecular mechanisms of genome functions requires identification of molecules interacting with genomic regions of interest in vivo. To this end, it is useful to isolate the target regions retaining molecular interactions. We established locus-specific chromatin immunoprecipitation (ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) for isolation of target genomic regions (Hoshino and Fujii, 2009; Fujita and Fujii, 2011; Fujita and Fujii, 2012; Fujita and Fujii, 2013a; Fujita and Fujii, 2013b; Fujita et al., 2013). Identification and characterization of molecules interacting with the isolated genomic regions facilitates understanding of molecular mechanisms of functions of the target genome regions. Here, we describe enChIP, in which engineered DNA-binding molecules, such as zinc-finger proteins, transcription activator-like (TAL) proteins, and a catalytically inactive Cas9 (dCas9) plus small guide RNA (gRNA), are utilized for affinity purification of target genomic regions. The scheme of enChIP is as follows: 1. A zinc-finger protein, TAL or dCas9 plus gRNA is generated to recognize DNA sequence in a genomic region of interest. 2. The engineered DNA-binding molecule is fused with a tag(s) and the nuclear localization signal (NLS), and expressed in the cell to be analyzed. 3. The resultant cell is crosslinked, if necessary, and lysed, and DNA is fragmented. 4. The complexes including the engineered DNA-binding molecule are subjected to affinity purification such as immunoprecipitation. The isolated complexes retain molecules interacting with the genomic region of interest. 5. Reverse crosslinking and subsequent purification of DNA, RNA, or proteins allow identification and characterization of these molecules. In this protocol, we describe enChIP with a TAL protein to isolate a genomic region of interest and analyze the interacting proteins by mass spectrometry (Fujita et al., 2013).