Direct isolation and identification of promoters in the human genome
Direct isolation and identification of promoters in the human genome
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DOI：
10.1101/gr.3430605
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发表时间：
2005-06-01
期刊：
影响因子：
7
通讯作者：
Ren, B
中科院分区：
文献类型：
--
作者：
Kim, TH;Barrera, LO;Ren, B
转录调控元件在动物发育过程中的基因表达和细胞对环境信号的反应中起着至关重要的作用，但是尽管有完整的基因组序列，我们对人类基因组中这些区域的了解还是有限的。启动子标志着每个转录本的开始，是一类重要的调控元件。所有活性启动子上都装配了一个大而复杂的蛋白质结构，称为起始前复合物（pre-initiation complex， PIC），这些蛋白质的存在将启动子与基因组中的其他序列区分开来。使用PIC的成分作为标签，我们直接从人类细胞中分离出启动子作为蛋白质-DNA复合物，并使用基因组平铺微阵列鉴定了得到的DNA序列。我们在4个人类细胞系中进行的实验发现，在44个半随机选择的人类基因组区域中发现了252个pic结合位点，这些区域占人类基因组的1%（30个碱基对）。近72%的已鉴定片段重叠或直接位于已知cDNA序列的5'端，而其余部分则位于其他基因组区域，可能含有未注释转录本的假定启动子。事实上，从一个细胞系中分离的RNA的分子分析发现了超过一半的假定启动子片段的转录本，瞬时转染实验显示，当它们与荧光素酶报告基因融合时，相当一部分片段的启动子活性很高。这些结果证明了全基因组分析方法定位转录调控元件的特异性，也表明还有少量但重要的人类基因有待发现。
Transcriptional regulatory elements play essential roles in gene expression during animal development and cellular response to environmental signals, but our knowledge of these regions in the human genome is limited despite the availability of the complete genome sequence. Promoters mark the start of every transcript and are an important class of regulatory elements. A large, complex protein structure known as the pre-initiation complex (PIC) is assembled on all active promoters, and the presence of these proteins distinguishes promoters from other sequences in the genome. Using components of the PIC as tags, we isolated promoters directly from human cells as protein-DNA complexes and identified the resulting DNA sequences using genomic tiling microarrays. Our experiments in four human cell lines uncovered 252 PIC-bincling sites in 44 semirandomly selected human genomic regions comprising 1% (30 me-abase pairs) of the human genome. Nearly 72% of the identified fragments overlap or immediately flank 5' ends of known cDNA sequences, while the remainder is found in other genomic regions that likely harbor putative promoters of unannotated transcripts. Indeed, molecular analysis of the RNA isolated from one cell line uncovered transcripts initiated from over half of the Putative promoter fragments, and transient transfection assays revealed promoter activity for a significant proportion of fragments when they were fused to a luciferase reporter gene. These results demonstrate the specificity of a genome-wide analysis method for mapping transcriptional regulatory elements and also indicate that a small, yet significant number of human genes remains to be discovered.