Chromatin Position Effects Assayed by Thousands of Reporters Integrated in Parallel
Chromatin Position Effects Assayed by Thousands of Reporters Integrated in Parallel
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DOI：
10.1016/j.cell.2013.07.018
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发表时间：
2013-08-15
期刊：
影响因子：
64.5
通讯作者：
van Steensel, Bas
中科院分区：
文献类型：
--
作者：
Akhtar, Waseem;de Jong, Johann;van Steensel, Bas
整合到基因组中的报告基因是揭示调控元件和局部染色质环境对基因表达影响的有力工具。然而，到目前为止，这种记者分析的吞吐量一直很低。在这里，我们描述了一种多路复用的方法，用于并行监测数千名随机整合的记者的转录活动。在用两种不同的启动子获得的小鼠胚胎干细胞中，超过27,000个不同的报告整合显示出类似于1,000倍表达水平的差异。数据分析表明，片层相关结构域作为转录衰减器，可能通过减少转录因子对结合位点的访问而发挥作用。此外，染色质压缩可以预测报告活性。我们还发现了相邻基因之间存在串扰的证据，并估计增强子对基因表达的影响平均类似于20kb。多重报告分析在设计上具有高度的灵活性，并且可以进行修改以询问基因调控的广泛方面。
Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two differentpromoters, show similar to 1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over similar to 20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation.