Chromatin position effects assayed by thousands of reporters integrated in parallel (mPGK TRIP pools)

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 different promoters, show ~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 cross-talk between neighboring genes, and estimate that enhancers can influence gene expression on average over ~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.

整合至基因组的报告基因（Reporter genes）是揭示调控元件与局部染色质环境对基因表达产生影响的强有力研究工具。然而迄今为止，此类报告基因检测（reporter assays）的通量普遍较低。本研究介绍一种可并行监测数千个随机整合报告基因转录活性的多重检测方法。我们利用两种不同的启动子（promoters）在小鼠胚胎干细胞（mouse embryonic stem cells）中获得了超过27000个独立的报告基因整合位点，其表达水平存在约1000倍的差异。数据分析结果显示，核纤层关联结构域（lamina-associated domains）可作为转录的衰减调控元件，其作用机制可能是通过降低转录因子（transcription factors）与其结合位点（binding sites）的接触几率。此外，染色质压缩程度可用于预测报告基因的表达活性。本研究还发现了邻近基因间存在串扰（cross-talk）的证据，并估算出增强子（enhancers）平均可对约20千碱基对（kb）范围内的基因表达产生调控作用。该多重报告基因检测方法设计灵活性极强，可通过改造用于探究基因调控领域的诸多方向。