Identification of active transcriptional regulatory modules by the functional assay of DNA from nucleosome-free regions.
Identification of active transcriptional regulatory modules by the functional assay of DNA from nucleosome-free regions.
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DOI：
10.1101/gr.073460.107
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发表时间：
2008-06
期刊：
影响因子：
7
通讯作者：
Mahesh Yaragatti;C. Basilico;L. Dailey
中科院分区：
文献类型：
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作者：
Mahesh Yaragatti;C. Basilico;L. Dailey
识别哺乳动物基因组中的转录调控模块是理解调控基因表达的机制的先决条件。虽然基于高通量微阵列和测序的方法已经被用来绘制特定蛋白质因子结合的核酸酶敏感部位或靶DNA序列的基因组位置图，但使用功能分析来识别调控元件，这将提供重要的补充数据，相对较少。在这里，我们提出了一种方法，允许使用一种简单的程序来分离和分析来自核小体无区(NFR)的DNA，该方法允许对活性转录调控模块进行功能鉴定。NFR是细胞基因组中包含这些元件的2%。以这种方式从F9细胞中鉴定出的100多个新的活性调控DNA对应于启动子近端和远端元件，并显示出几个预测的内源性转录调控因子的特征，包括定位在DNA酶可访问的染色质和CpG岛上，以及接近表达的基因。此外，与已发表的ES细胞染色质CHIP-SEQ数据比较表明，我们确定的功能元件对应于富含H3K4me3的基因组区域，H3K4me3是一种与活性转录调节元件相关的组蛋白修饰，并且H3K4me3与我们的启动子-远端元件的对应关系在很大程度上是ES细胞特有的。大多数远端元件显示出增强活性。重要的是，这些功能DNA片段的平均长度为149bp，这极大地促进了未来的应用，以确定介导其活性的转录因子结合位点。因此，这种方法为高分辨率鉴定活性启动子和增强子的功能成分提供了一种工具。
The identification of transcriptional regulatory modules within mammalian genomes is a prerequisite to understanding the mechanisms controlling regulated gene expression. While high-throughput microarray- and sequencing-based approaches have been used to map the genomic locations of sites of nuclease hypersensitivity or target DNA sequences bound by specific protein factors, the identification of regulatory elements using functional assays, which would provide important complementary data, has been relatively rare. Here we present a method that permits the functional identification of active transcriptional regulatory modules using a simple procedure for the isolation and analysis of DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular genome that contains these elements. The more than 100 new active regulatory DNAs identified in this manner from F9 cells correspond to both promoter-proximal and distal elements, and display several features predicted for endogenous transcriptional regulators, including localization within DNase-accessible chromatin and CpG islands, and proximity to expressed genes. Furthermore, comparison with published ChIP-seq data of ES-cell chromatin shows that the functional elements we identified correspond with genomic regions enriched for H3K4me3, a histone modification associated with active transcriptional regulatory elements, and that the correspondence of H3K4me3 with our promoter-distal elements is largely ES-cell specific. The majority of the distal elements exhibit enhancer activity. Importantly, these functional DNA fragments are an average 149 bp in length, greatly facilitating future applications to identify transcription factor binding sites mediating their activity. Thus, this approach provides a tool for the high-resolution identification of the functional components of active promoters and enhancers.