Mapping cis- and trans- chromatin interaction networks using chromosome conformation capture (3C).
Mapping cis- and trans- chromatin interaction networks using chromosome conformation capture (3C).
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DOI：
10.1007/978-1-60327-461-6_7
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Dekker, Job
中科院分区：
文献类型：
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作者：
Miele, Adriana;Dekker, Job
染色体在细胞内以复杂的方式折叠，它们的空间组织与基因表达的调控密切相关。基因的表达可以由位于距其靶基因较大基因组距离处（顺式）或甚至位于不同染色体上（反式）的调控元件控制。调控元件可以通过与其靶基因直接物理相互作用而在大的基因组距离处起作用，从而导致染色质环的形成。因此，基因及其调控元件在空间上非常接近，而不管它们的相对基因组位置如何。对基因和元件之间相互作用的分析将揭示哪些元件调节每个基因，并将为一般染色体的空间组织提供基本见解。可以使用染色体构象捕获（3C）技术以高分辨率研究长程顺式和反式相互作用。3C采用甲醛交联来捕获位于整个基因组中的基因座之间的物理相互作用。然后溶解交联的细胞，并通过限制酶消化染色质。消化后，染色质在非常稀的DNA浓度下进行连接。这些条件有利于分子内连接而不是分子间连接，因此导致相互作用（和交联）的基因组元件的选择性连接。交联被逆转，DNA被纯化，并且特定染色体基因座之间的相互作用频率可以通过定量使用PCR形成的相应连接产物的量来确定。本章描述了酵母酿酒酵母和哺乳动物染色体的3C分析的详细协议。
Chromosomes are folded in intricate ways inside cells and their spatial organization is intimately related to regulation of gene expression. Expression of genes can be controlled by regulatory elements that are located at large genomic distances from their target genes (in cis), or even on different chromosomes (in trans). Regulatory elements can act at large genomic distances by engaging in direct physical interactions with their target genes resulting in the formation of chromatin loops. Thus, genes and their regulatory elements come in close spatial proximity irrespective of their relative genomic positions. Analysis of interactions between genes and elements will reveal which elements regulate each gene, and will provide fundamental insights into the spatial organization of chromosomes in general. Long-range cis- and trans- interactions can be studied at high resolution using the Chromosome Conformation Capture (3C) technology. 3C employs formaldehyde crosslinking to trap physical interactions between loci located throughout the genome. Crosslinked cells are then solubilized and chromatin is digested by a restriction enzyme. After digestion the chromatin is subjected to ligation under very dilute DNA concentrations. These conditions favor intramolecular ligation over intermolecular ligation, and thus result in selective ligation of interacting (and crosslinked) genomic elements. The crosslinks are reversed, the DNA is purified, and interaction frequencies between specific chromosomal loci can be determined by quantifying the amounts of corresponding ligation product that is formed using PCR. This chapter describes detailed protocols for 3C analysis of yeast Saccharomyces cerevisiae and mammalian chromosomes.