Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish “insulator-seq” as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators.

中文翻译：

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果蝇中增强子阻断绝缘子的系统筛选确定了它们的 DNA 序列决定因素


动物基因组中丰富的增强子对基因启动子的长程转录激活需要限制不适当调节的机制。称为绝缘体的 DNA 元件通过在插入时保护启动子免受增强子的影响来达到此目的。与启动子和增强子不同，由于缺乏高通量筛选测定，绝缘子尚未得到系统的表征，并且有关绝缘子如何在基因组中分布和编码的问题仍然存在。在这里，我们在果蝇培养的细胞中将“insulator-seq”建立为基于质粒的大规模平行报告基因测定，以对选定的基因组位点进行系统的绝缘子筛选。筛选发育基因位点表明，并非所有绝缘体蛋白结合位点都能有效阻断增强子-启动子通讯。深度绝缘体诱变鉴定了灵活定位在 CTCF 绝缘体蛋白结合基序周围的序列，这些序列对功能至关重要。在没有位置效应的情况下筛选数百万个 DNA 序列的能力使绝缘体的功能映射成为可能，并提供了对绝缘体决定因素的进一步见解。