Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae
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DOI：
10.3791/55836
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发表时间：
2017-06-01
影响因子：
1.2
通讯作者：
Zentner, Gabriel E.
中科院分区：
文献类型：
--
作者：
Grunberg, Sebastian;Zentner, Gabriel E.
蛋白质-DNA相互作用的全基因组图谱对于理解基因调控、染色质重塑和其他染色质驻留过程至关重要。甲醛交联，然后染色质免疫沉淀和高通量测序（X-ChIP-seq）已被用来获得许多有价值的见解基因组生物学。然而，X-ChIP-seq具有与交联和超声处理相关的显著限制。天然ChIP通过省略交联避免了这些缺点，但通常导致染色质结合蛋白的回收率差。此外，所有基于ChIP的方法都需要考虑抗体质量。用于绘制蛋白质-DNA相互作用的酶促方法，其涉及将感兴趣的蛋白质与DNA修饰酶融合，也已用于绘制蛋白质-DNA相互作用。我们最近将一种这样的方法，染色质内源性切割（ChEC），与高通量测序相结合，称为ChEC-seq。ChEC-seq依赖于目的染色质相关蛋白与微球菌核酸酶（MNase）的融合，以在活细胞中存在钙的情况下产生靶向DNA切割。ChEC-seq不基于免疫沉淀，因此避免了交联、超声处理、染色质溶解和抗体质量的潜在问题，同时提供具有最小背景信号的高分辨率作图。我们设想ChEC-seq将成为ChIP的强大对应物，提供一种独立的方法来验证ChIP-seq发现并发现基因组调控的新见解。
Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatinresident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.