Using ChIP-seq technology to identify targets of zinc finger transcription factors.
Using ChIP-seq technology to identify targets of zinc finger transcription factors.
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DOI：
10.1007/978-1-60761-753-2_27
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Farnham, Peggy J
中科院分区：
文献类型：
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作者：
O'Geen, Henriette;Frietze, Seth;Farnham, Peggy J
一半的人类转录因子是锌指蛋白，但对大多数这些因子的生物学作用知之甚少。特别是，很少进行锌指因子体内结合的全基因组研究。基于体外研究和其他允许在人工条件下选择高亲和力结合位点的方法，锌指编码已被开发出来，可用于构建特定锌指因子（ZNF）的推定识别基序。从理论上讲，一个简单的生物信息学分析就可以预测ZNF所有结合位点的基因组位置。然而，人类基因组中与预测基序具有良好匹配的所有序列在体内实际上不太可能被占据（由于抑制染色质、核小体定位、与其他因素结合位点重叠等负面影响）。在全基因组范围内鉴定转录因子体内结合位点的一种有效方法是染色质免疫沉淀（ChIP）测定，然后将沉淀的DNA与微阵列（ChIP- ChIP）杂交或对样品进行高通量DNA测序（ChIP-seq）。这种全面的体内结合研究不仅可以识别特定锌指因子的靶基因，还可以提供结合基元数据，用于测试锌指编码的有效性。本章详细描述了使用Illumina GA2平台制备高通量测序所需的ChIP样品和文库的步骤，并包括确保ChIP-seq实验成功所需的质量控制步骤的描述。
Half of all human transcription factors are zinc finger proteins and yet very little is known concerning the biological role of the majority of these factors. In particular, very few genome-wide studies of the in vivo binding of zinc finger factors have been performed. Based on in vitro studies and other methods that allow selection of high affinity-binding sites in artificial conditions, a zinc finger code has been developed that can be used to compose a putative recognition motif for a particular zinc finger factor (ZNF). Theoretically, a simple bioinformatics analysis could then predict the genomic locations of all the binding sites for that ZNF. However, it is unlikely that all of the sequences in the human genome having a good match to a predicted motif are in fact occupied in vivo (due to negative influences from repressive chromatin, nucleosomal positioning, overlap of binding sites with other factors, etc). A powerful method to identify in vivo binding sites for transcription factors on a genome-wide scale is the chromatin immunoprecipitation (ChIP) assay, followed by hybridization of the precipitated DNA to microarrays (ChIP-chip) or by high throughput DNA sequencing of the sample (ChIP-seq). Such comprehensive in vivo binding studies would not only identify target genes of a particular zinc finger factor, but also provide binding motif data that could be used to test the validity of the zinc finger code. This chapter describes in detail the steps needed to prepare ChIP samples and libraries for high throughput sequencing using the Illumina GA2 platform and includes descriptions of quality control steps necessary to ensure a successful ChIP-seq experiment.