Background Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics. Results Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in “ground-state” (2i+LIF; 2iL) and “metastable” (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency. Conclusions In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

中文翻译：

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STARR-seq 鉴定多能小鼠胚胎干细胞中的活性增强子、染色质掩蔽增强子和休眠增强子


背景增强子是基因表达的远端调节因子，塑造细胞身份并控制细胞命运转变。在小鼠胚胎干细胞（mESC）中，多能性网络是通过复杂的增强子网络的功能来维持的，增强子网络在分化时会发生巨大的改变。全基因组染色质可及性和组蛋白修饰测定通常用作识别假定增强子并描述其活性水平和动态的代理。结果在这里，我们应用 STARR-seq（一种基于质粒的全基因组检测）作为“基态”（2i+LIF；2iL）和“亚稳态”（血清+LIF；SL）增强子景观的读数) mESC。该分析表明，活性 STARR-seq 位点与从 ChIP-seq 文库的常见增强子标记的峰调用得出的增强子位置显示出适度的重叠。我们揭示了 SL-ESC 中具有显着 STARR-seq 活性的 ZIC3 结合位点。 Zic3 的敲除仅消除 SL-ESC 中的 STARR-seq 活性，并增加其分化为内胚层命运的倾向。 STARR-seq 还揭示了不可访问的增强子，这些增强子被抑制性染色质特征所掩盖。我们描述了一类休眠的 p53 结合增强子，它们在特定条件下获得 H3K27ac，例如用 Nocodazol 治疗后，或在从成纤维细胞重编程为多能性期间短暂地获得 H3K27ac。结论 总之，STARR-seq 鉴定为活性的基因座通常与染色质可及性和活性表观遗传标记鉴定的基因座重叠，但很大一部分受到表观遗传抑制或表现出条件特异性增强子活性。