Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation
Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation
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DOI：
10.2144/000112014
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发表时间：
2005-11-01
期刊：
影响因子：
2.7
通讯作者：
Brasier, AR
中科院分区：
文献类型：
--
作者：
Nowak, DE;Tian, B;Brasier, AR
染色质免疫沉淀（ChIP）测定最近已被开发为一个强大的和通用的技术，用于探测染色质环境中的蛋白质-DNA相互作用。在该方法中，用可逆DNA-蛋白质交联剂（甲醛）固定完整细胞，并通过免疫沉淀靶DNA结合蛋白来富集相关DNA。然后，免疫复合物中结合的DNA用于鉴定特异性DNA结合蛋白的内源性基因组靶标。核因子κ B（NF-κ B）是一种高度诱导的转录因子，其控制对于病原体或病毒诱导的炎症、免疫应答和细胞存活重要的遗传网络。在我们对诱导型NF-κ B转录因子控制下的遗传网络的研究中，我们发现使用单一甲醛交联步骤的常规ChIP技术不能将其可重复地交联到DNA上。因此，我们已经开发了一种新的ChIP检测使用两步交联程序，将N-羟基琥珀酰亚胺（NHS）-酯介导的蛋白质-蛋白质交联之前，常规的DNA-蛋白质交联。我们证明了这种技术是高效的，几乎所有的NF-κ B/Rel A交联成共价复合物，导致可诱导的NF-κ B家族与多种经验证的NF-κ B依赖性基因组靶点结合的定量和稳健鉴定。为了证明这种两步交联程序的一般效用，我们进行了增强的捕获的精氨酸诱导的信号转导和转录激活因子-3（STAT 3）结合到其已知的靶基因之一。我们的方法代表了一个显着的改善，在研究罕见的转录因子的内源性目标的ChIP分析的效率。
The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this method, intact cells are fixed with a reversible DNA-protein cross-linking agent (formaldehyde), and associated DNA is enriched by immunoprecipitating a target DNA binding protein. The bound DNA in the immune complexes is then used to identify that specific DNA binding protein's endogenous genomic targets. Nuclear factor kappa B (NF-kappa B) is a highly inducible transcription factor that controls genetic networks important for pathogen- or cytokine-induced inflammation, immune response, and cellular survival. In our studies of the genetic network under control of the inducible NF-kappa B transcription factor we found that the conventional ChIP technique using a single formaldehyde cross-linking step did not reproducibly cross-link it to DNA. As a result, we have developed a novel ChIP assay using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross-linking prior to conventional DNA-protein cross-linking. We demonstrate that this technique is highly efficient, cross-linking virtually all NF-kappa B/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-kappa B family binding to a variety of validated NF-kappa B-dependent genomic targets. To demonstrate the general utility of this two-step cross-linking procedure, we performed enhanced capture of cytokine-inducible signal transducer and activator of transcription-3 (STAT3) binding to one of its known target genes. Our method represents a significant improvement in the efficiency of ChIP analysis in the study of endogenous targets for rare transcription factors.