In vivo enhancer analysis of human conserved non-coding sequences
In vivo enhancer analysis of human conserved non-coding sequences
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DOI：
10.1038/nature05295
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发表时间：
2006-11-23
期刊：
影响因子：
64.8
通讯作者：
Rubin, Edward M.
中科院分区：
文献类型：
--
作者：
Pennacchio, Len A.;Ahituv, Nadav;Rubin, Edward M.
鉴定指导基因的空间和时间表达的序列并定义它们在体内的功能仍然是脊椎动物基因组注释中的一个重大挑战。一个主要的障碍是缺乏经过实验验证的训练集。在这项研究中，我们利用极端进化序列保守性作为筛选器来鉴定推定的基因调控元件，并表征了人类基因组中一大组非编码元件的体内增强子活性，这些元件在人类-河豚、红鳍东方鲀（东方鲀）中保守，或在人类-小鼠-大鼠中超保守（1）。我们在转基因小鼠增强子试验中测试了167个这些极其保守的序列。在这里，我们报告说，这些序列中的45%在胚胎第11.5天可重复地作为组织特异性基因表达增强子发挥作用。虽然在胚胎的广泛解剖结构中指导表达，但75个增强子中的大多数将表达定向到发育中的神经系统的各个区域。我们鉴定了富含这些元件的一个子集的序列特征，这些元件靶向前脑表达，并使用这些特征对人类基因组中在人类和河豚之间保守的所有3,100个非编码元件进行排名。在转基因小鼠中对最高预测的测试导致具有前脑增强子活性的序列的三倍富集。这些数据极大地扩展了已在体内表征的人类基因增强子的目录，并说明了这种训练集在各种生物学应用中的实用性，包括解码人类基因组的调控词汇。
Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultra-conserved(1) in human-mouse-rat. Wetested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.