Tian Bing, Yang Jun, Brasier Allan R
Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.
Methods Mol Biol. 2012;809:105-20. doi: 10.1007/978-1-61779-376-9_7.
Eukaryotic gene regulation is controlled, in part, by inducible transcription factor-binding regulatory sequences in a tissue-specific and hormone-responsive manner. The development of methods for the analysis of transcription factor interaction within native chromatin has been a significant advance for the systematic analyses of the timing of gene regulation and studies on the effects of chromatin modifying enzymes on promoter accessibility. Chromatin immunoprecipitation (ChIP) is a specific method involving formaldehyde mediated protein-chromatin fixation to preserve the interaction for subsequent target identification. However, the conventional single-step cross-linking technique does not preserve all protein-DNA interactions, especially for transcription factors in hyper-dynamic equilibrium with chromatin or for coactivator interactions. Here, we describe a versatile, efficient "two-step" XChIP method that involves sequential protein-protein fixation followed by protein-DNA fixation. This method has been used successfully for analysis of chromatin binding for transcription factors (NF-κB, STAT3), polymerases (RNA Pol II), coactivators (CBP/p300, CDK9), and chromatin structural proteins (modified histones). Modifications of DNA extraction and sonication suitable for downstream target identification by quantitative genomic PCR and next generation sequencing are described.
真核基因调控部分是通过以组织特异性和激素应答方式的诱导型转录因子结合调控序列来控制的。用于分析天然染色质中转录因子相互作用的方法的发展，对于系统分析基因调控的时间以及研究染色质修饰酶对启动子可及性的影响而言，是一项重大进展。染色质免疫沉淀（ChIP）是一种特定方法，它涉及甲醛介导的蛋白质 - 染色质固定，以保留相互作用以便后续进行靶点鉴定。然而，传统的单步交联技术并不能保留所有的蛋白质 - DNA相互作用，特别是对于与染色质处于超动态平衡的转录因子或共激活因子相互作用而言。在此，我们描述了一种通用、高效的“两步”XChIP方法，该方法涉及先后进行蛋白质 - 蛋白质固定和蛋白质 - DNA固定。此方法已成功用于分析转录因子（NF - κB、STAT3）、聚合酶（RNA Pol II）、共激活因子（CBP/p300、CDK9）以及染色质结构蛋白（修饰组蛋白）与染色质的结合。文中还描述了适用于通过定量基因组PCR和下一代测序进行下游靶点鉴定的DNA提取和超声处理的改进方法。
