SnapShot: Chromosome confirmation capture.
SnapShot: Chromosome confirmation capture.
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DOI：
10.1016/j.cell.2012.02.019
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发表时间：
2012-03-02
期刊：
影响因子：
64.5
通讯作者：
Misteli T
中科院分区：
文献类型：
--
作者：
Hakim O;Misteli T
基因组在核空间中的组织是非随机的，并影响基因组的功能，包括转录、复制和修复。来自相同或不同染色体的特定基因组区域经常相互联系并与细胞核结构联系在一起，从而产生错综复杂的区隔细胞核。基因组相互作用的例子是增强子与启动子的关联或核仁中rDNA基因等基因的聚类。基因组相互作用传统上是用荧光原位杂交（FISH）来研究的，它可以可视化不同基因或基因组区域之间的空间关系。这种方法的局限性是，只能询问已知的相互作用，在实验中只能探测很少的位点，并且分辨率仅限于显微镜的光学。染色体家族构象捕获技术是一套确定基因组区域物理相互作用的生化方法。c -技术方法总是涉及五个步骤：(1)甲醛固定在物理相互作用位点交联染色质；(2)用限制性内切酶或超声裂解染色质；(3)在稀释条件下的连接，更倾向于在同一复合体上捕获的DNA末端之间的连接，而不是随机碰撞的连接；(4)根据方法的不同，使用可变的分子生物学步骤检测连接连接。(5)计算分析，以确定在交联染色质连接中捕获的相互作用频率。c -技术（3C, 4C, 5C, Hi-C）在检测方式和可以探测的相互作用范围方面有所不同。3C方法测试基因组中两个已知位点之间的相互作用，4C方法允许探测已知诱饵序列的未知相互作用，5C方法识别给定基因组域中的所有相互作用区域，Hi-C方法以无偏的方式在全基因组范围内探测所有发生的相互作用。其他变体（ChIA-PET, ChIP-Loop）包含蛋白质沉淀步骤，允许鉴定涉及特定感兴趣蛋白质的基因组相互作用。方法的选择在很大程度上取决于生物学问题的具体性质和范围，但也取决于资源的可得性，包括起始材料的数量和测序能力。标准c -技术的许多衍生品已经开发出来，通常受到特定生物学问题的启发，或以提高特异性或减少背景为目标。c技术是基于人群的方法。它们产生的是相对接触概率，而不是绝对接触频率。基于群体的性质是由于每个基因组位点在一个细胞中提供一个成对的结扎连接。为了实现接触谱的高覆盖率和定量评估，必须在每个实验中包括并组合包含多个连接连接的数千到数百万个基因组当量（细胞）。C接触与DNA FISH之间的相关性表明，在大多数C方法中，在人群中3%-5%的细胞中发生的染色体间关联通常被检测为阳性。更频繁的联想通常会产生更强的信号；然而，信号的强度也可能反映物理相互作用的亲和力，而不是它的频率。数据分析中的一个关键步骤是确定作为结扎结检测到的相互作用是否具有特异性。接触频率呈指数下降，并与距离参考点几Mb的线性基因组距离成反比。因此，……
The organization of the genome in the nuclear space is nonrandom and affects genome functions, including transcription, replication, and repair. Specific genomic regions, from the same or different chromosomes, frequently physically associate with each other and with nuclear structures, giving rise to an intricately compartmentalized nucleus. Examples of genome interactions are the association of an enhancer with a promoter or the clustering of genes such as rDNA genes in the nucleolus. Genome interactions have traditionally been studied using fluorescence in situ hybridization (FISH), which allows visualization of the spatial relationship between distinct genes or genome regions. Limitations of this method are that only known interactions can be interrogated, only very few loci can be probed in an experiment, and resolution is limited to the optics of the microscope. The family of chromosome conformation capture techniques is a set of biochemical approaches to determine the physical interaction of genome regions. C-technology approaches invariably involve five steps:(1) formaldehyde fixation to crosslink chromatin at sites of physical interaction,(2) cleavage of chromatin by restriction enzyme or sonication,(3) ligation under dilute conditions favoring ligation between DNA ends captured on the same complex over ligations from random collisions,(4) detection of ligation junctions using variable molecular biology steps depending on the variant of the methods, and (5) computational analysis to determine interaction frequencies captured in the ligation of the crosslinked chromatin.C-technologies (3C, 4C, 5C, Hi-C) differ in their manner of detection and scope of what interactions they can probe. The 3C method tests the interaction between two known sites in the genome, 4C allows probing of unknown interactors of a known bait sequence, 5C identifies all regions of interaction within a given genome domain, and Hi-C probes all occurring interactions in an unbiased fashion genome-wide. Additional variants (ChIA-PET, ChIP-Loop) incorporate a protein precipitation step, allowing identification of genome interactions that involve a specific protein of interest. The choice of method strongly depends on the specific nature and scope of the biological question, but also on the availability of resources, including the amount of starting material and sequencing capacity. Many derivatives of the standard C-techniques have been developed, often inspired by the specific biological question addressed or with the goal of improving specificity or reducing background. C-technologies are population-based methods. They produce relative contact probabilities rather than absolute contact frequencies. The population-based nature is due to the fact that each genomic locus gives one pair-wise ligation junction in one cell. To allow high coverage and quantitative appraisal of contact profiles, thousands to millions of genome equivalents (cells) containing multiple ligation junctions must be included and combined in each experiment. Correlations between C contacts and DNA FISH have indicated that an interchromosomal association that occurs in 3%–5% of cells in a population will typically be detected as positive in most C methods. More frequent associations generally result in stronger signals; however, the strength of signal may also reflect the affinity of the physical interactions and not its frequency. A critical step in data analysis is to determine whether an interaction, detected as a ligation junction, is specific. The contact frequency decreases exponentially and is inversely related to the linear genomic distance up to a few Mb away from the reference point. Therefore, the …
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