Using ChIP-chip and ChIP-seq to study the regulation of gene expression: genome-wide localization studies reveal widespread regulation of transcription elongation.
Using ChIP-chip and ChIP-seq to study the regulation of gene expression: genome-wide localization studies reveal widespread regulation of transcription elongation.
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DOI：
10.1016/j.ymeth.2009.02.024
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发表时间：
2009-08
期刊：
影响因子：
4.8
通讯作者：
Adelman, Karen
中科院分区：
文献类型：
--
作者：
Gilchrist, Daniel A.;Fargo, David C.;Adelman, Karen
转录是一个复杂的多步骤过程，其中RNA聚合酶II（Pol II）与广泛的转录起始、延伸、加帽、终止和组蛋白修饰因子一起将DNA模板转录成RNA。最近的全球分析Pol II分布表明，许多基因在伸长阶段受到调控，揭示了以前未被充分认识的控制基因表达的机制。染色质免疫沉淀（ChIP）研究极大地帮助了了解各种因素如何调节活细胞中的转录延伸，该研究可以提供蛋白质-DNA结合事件的空间和时间分辨率。ChIP与DNA微阵列和高通量测序技术（ChIP-chip和ChIP-seq）的结合显着增加了ChIP研究的范围，现在可以很容易地产生Pol II或延伸因子结合位点的全基因组图谱。然而，虽然ChIP-chip/ChIP-seq数据允许蛋白质-DNA结合位点的高分辨率定位，但它们不足以剖析蛋白质功能。在这里，我们描述了将ChIP-chip/ChIP-seq与遗传、化学和实验操作相结合的技术，以从全基因组蛋白质-DNA结合研究中获得机理见解。我们已经采用这些技术来辨别未成熟的启动子近端Pol II从生产性延长Pol II，并推断在调节发育和基因诱导响应环境信号的启动和完全延伸能力之间的过渡的关键作用。
Transcription is a sophisticated multi-step process in which RNA polymerase II (Pol II) transcribes a DNA template into RNA in concert with a broad array of transcription initiation, elongation, capping, termination, and histone modifying factors. Recent global analyses of Pol II distribution have indicated that many genes are regulated during the elongation phase, shedding light on a previously underappreciated mechanism for controlling gene expression. Understanding how various factors regulate transcription elongation in living cells has been greatly aided by chromatin immunoprecipitation (ChIP) studies, which can provide spatial and temporal resolution of protein-DNA binding events. The coupling of ChIP with DNA microarray and high-throughput sequencing technologies (ChIP-chip and ChIP-seq) has significantly increased the scope of ChIP studies and genome-wide maps of Pol II or elongation factor binding sites can now be readily produced. However, while ChIP-chip/ChIP-seq data allow for high-resolution localization of protein-DNA binding sites, they are not sufficient to dissect protein function. Here we describe techniques for coupling ChIP-chip/ChIP-seq with genetic, chemical, and experimental manipulation to obtain mechanistic insight from genome-wide protein-DNA binding studies. We have employed these techniques to discern immature promoter-proximal Pol II from productively elongating Pol II, and infer a critical role for the transition between initiation and full elongation competence in regulating development and gene induction in response to environmental signals.
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