Massively parallel characterization of insulator activity across the genome [insulator_libraries]

Insulators are cis-regulatory sequences (CRSs) that can block enhancers from activating target promoters or act as barriers to block the spread of heterochromatin. Their name derives from their ability to 'insulate' transgenes from genomic position effects, an important function in gene therapy and biotechnology applications that require high levels of sustained transgene expression. In theory, flanking transgenes with insulators protects them from position effects, but in practice, efforts to insulate transgenes meet with mixed success because the contextual requirements for insulator function in the genome are not well understood. A key question is whether insulators are modular elements that can function anywhere in the genome or whether they are adapted to function only in certain genomic locations. To distinguish between these two possibilities we developed MPIRE (Massively Parallel Integrated Regulatory Elements) and used it to measure the effects of three insulators (A2, cHS4, ALOXE3) and their mutants at thousands of locations across the genome. Our results show that each insulator functions in only a small number of genomic locations, and that insulator function depends on the sequence motifs that comprise each insulator. All three insulators can block enhancers in the genome, but specificity arises because each insulator blocks enhancers that are bound by different sets of transcription factors. In contrast, only ALOXE3 can act as a heterochromatin barrier. We conclude that insulator function is highly context dependent and that MPIRE is a robust and systematic method for revealing the context dependencies of insulators and other cis-regulatory elements across the genome. Overall design: Barocde sequencing from genome-integrated massively parallel reporter assays

绝缘子是一类顺式调控序列（cis-regulatory sequences, CRSs），能够阻断增强子激活靶启动子，或作为屏障阻断异染色质的扩散。其命名源于其可将转基因与基因组位置效应相“绝缘”的能力——这一功能在需要高水平持续转基因表达的基因治疗与生物技术应用中至关重要。理论上，在转基因两侧侧翼加装绝缘子可使其免受位置效应影响，但实际操作中，这类绝缘子化转基因的尝试效果参差不齐，原因是我们对绝缘子在基因组中发挥功能的背景条件尚缺乏充分认知。核心问题在于，绝缘子究竟是可在基因组任意位置发挥功能的模块化元件，还是仅能在特定基因组区域行使功能的序列。为区分这两种可能性，我们开发了大规模并行整合调控元件（Massively Parallel Integrated Regulatory Elements, MPIRE）技术，并利用该技术在全基因组数千个位点上，检测了三种绝缘子（A2、cHS4、ALOXE3）及其突变体的功能效应。研究结果显示，每种绝缘子仅能在少量基因组位点发挥功能，且绝缘子的功能依赖于构成其序列的基序。三种绝缘子均可阻断基因组内的增强子，但特异性源于不同绝缘子可阻断结合不同转录因子组合的增强子。与之不同的是，仅ALOXE3可发挥异染色质屏障的功能。我们由此得出结论：绝缘子的功能具有高度的背景依赖性，而MPIRE是一种可靠且系统化的方法，可用于揭示绝缘子及其他顺式调控元件在全基因组范围内的背景依赖特性。实验整体设计：基于基因组整合型大规模平行报告基因检测法的条形码测序。