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. Overall design: TRIP assay of mPGK promoter; 6 experiments, each with 2 technical replicates.

整合至基因组的报告基因（Reporter gene）是揭示调控元件与局部染色质环境对基因表达影响的强效研究工具。然而截至目前，此类报告基因检测技术的通量普遍偏低。本研究报道了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。我们通过两种不同启动子在小鼠胚胎干细胞中构建了超过27000个独立的报告基因整合株系，其表达水平呈现出约1000倍的差异。数据分析显示，核纤层关联结构域（lamina-associated domain, LAD）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与结合位点的结合可达性。此外，染色质压缩状态可有效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的证据，并估算出增强子可在平均约20千碱基对的范围内影响基因表达。该多重报告基因检测方法设计灵活性极高，可通过改造用于探究基因调控的诸多不同方面。整体实验设计：针对mPGK启动子的TRIP检测；共6组实验，每组均设置2次技术重复。