Buisine Nicolas, Ruan Xiaoan, Ruan Yijun, Sachs Laurent M
Function and Mechanism of Action of Thyroid Hormone Receptor Group, UMR 7221 CNRS and Muséum National d'Histoire Naturelle, Sorbonne Universités, Paris 75005, France.
The Jackson Laboratory of Genomic Medicine, Farmington, Connecticut 06030.
Cold Spring Harb Protoc. 2018 Aug 1;2018(8):pdb.prot097725. doi: 10.1101/pdb.prot097725.
Proper gene expression involves communication between the regulatory elements and promoters of genes. Because regulatory elements can be located over a large range of genomic distances (from as close as a few hundred bp to as much as several Mb away), contact and communication between regulators and the core transcriptional machinery at promoters are mediated through DNA looping. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing, is a leading high-throughput method for detection of genome wide chromatin interactions. Briefly, the method involves cross-linkage of chromatin (-DNA) fibers in cells in situ, fragmentation of the fixed chromatin-DNA complexes by sonication, followed by enrichment of the chromatin complexes with a dedicated antibody through the process of immunoprecipitation (IP). Next, application of the ChIA-PET protocol followed by deep sequencing and mapping of reads to the reference genome reveals both binding sites and remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIP sample preparation and can be completed in ∼5 d. The ChIA-PET method is detailed in an associated protocol. Because not all chromatin immunoprecipitation protocols are suitable for ChIA-PET, it is important to strictly follow this procedure before performing the ChIA-PET protocol.
正确的基因表达涉及基因的调控元件与启动子之间的相互作用。由于调控元件可位于基因组的较大距离范围内（近至几百个碱基对，远至数百万碱基对），调控因子与启动子处的核心转录机制之间的接触和交流是通过DNA环化介导的。如今，基于染色体构象捕获（3C）的方法能够有效地探测细胞核中的染色体折叠情况，从而对增强子与其靶启动子之间的物理邻近性进行分子描述。其中一种方法，即使用双末端标签（ChIA-PET）测序的染色质相互作用分析，是检测全基因组染色质相互作用的领先高通量方法。简而言之，该方法包括对细胞内的染色质（-DNA）纤维进行原位交联，通过超声处理将固定的染色质-DNA复合物片段化，然后通过免疫沉淀（IP）过程用特异性抗体富集染色质复合物。接下来，应用ChIA-PET方案，随后进行深度测序并将读数映射到参考基因组，可揭示由感兴趣的蛋白质因子介导的结合位点和远程染色质相互作用。此处详述的方法侧重于ChIP样品制备，大约5天即可完成。ChIA-PET方法在相关方案中有详细说明。由于并非所有的染色质免疫沉淀方案都适用于ChIA-PET，因此在执行ChIA-PET方案之前严格遵循此程序非常重要。
