Genomic context sensitizes regulatory elements to genetic disruption [CUT&RUN]. Genomic context sensitizes regulatory elements to genetic disruption [CUT&RUN]

Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis, thus it remains unclear to what extent their function is influenced by surrounding genomic context. Using our Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the Igf2/H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2/H19 locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting the activity of payloads delivered to the endogenous locus or to a safe harbor locus (Hprt) revealed that the functional elements comprising the Igf2/H19 locus are highly sensitive to their native context. Exchanging components of the Igf2/H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster in particular functioned poorly out of context, and required its native surroundings to activate Sox2 expression. Conversely, the Sox2 locus control region (LCR) could activate both Igf2 and H19 outside its native context, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across different cell types revealed that, while the H19 enhancers are tightly coordinated within their native locus, the Sox2 LCR acts more independently. We show that these enhancer clusters typify broader classes of loci genome-wide. Our results show that unexpected dependencies may influence even the most studied functional elements, and our synthetic regulatory genomics approach permits large-scale manipulation of complete loci to investigate how locus architecture relates to function. Overall design: CTCF CUT&RUN of mouse embryonic stem cells (mESCs) and mesendodermal cells differentiated using CHIR. CTCF CUT&RUN on 6 samples with different configurations of CTCF recognition sequences at the Igf2/H19 ICR

增强子功能的研究常采用截短型报告基因检测或单基因缺失分析等碎片化手段，因此目前仍不清楚其功能在多大程度上受周围基因组背景的影响。本研究利用靶向整合大片段DNA的Big-IN技术，分析了作为增强子选择性调控经典模型的Igf2/H19基因座的调控架构。本研究构建了包含157 kb功能性Igf2/H19基因座的搭载片段，并通过工程化突变在印记控制区（imprinting control region, ICR）基因层面调控CTCF结合，从而切换H19增强子簇的靶基因。通过对比递送至内源基因座与安全港基因座（safe harbor locus, Hprt）的搭载片段的活性，研究发现构成Igf2/H19基因座的功能元件对其原生基因组环境具有高度敏感性。将Igf2/H19基因座的元件与研究较为透彻的Sox2基因座进行交换后发现，H19增强子簇尤其难以脱离原生环境发挥功能，其激活Sox2表达依赖于自身原生的基因组环境。与之相反，Sox2基因座控制区（locus control region, LCR）可在脱离原生环境的情况下激活Igf2与H19的表达，但其活性仅受ICR处CTCF结合的部分调控。对不同细胞类型中的调控DNA激活效应进行分析后发现：尽管H19增强子在其原生基因座内呈现紧密协同的调控模式，但Sox2 LCR的作用更为独立。本研究证实，这类增强子簇代表了全基因组范围内更广范围的基因座类别。本研究结果表明，即便对研究最为深入的功能元件，也可能存在未被发现的依赖关系影响其功能；而我们的合成调控基因组学方法可实现完整基因座的大规模操作，从而探究基因座架构与其功能之间的关联。实验整体设计：对小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）以及使用CHIR诱导分化得到的中内胚层细胞开展CTCF CUT&RUN实验；同时对6种在Igf2/H19 ICR处带有不同CTCF识别序列配置的样本开展CTCF CUT&RUN实验。