Genomic context sensitizes regulatory elements to genetic disruption [DNAse-seq]

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: DNase-seq mapping in engineered mesendodermal cells differentiated from mouse embryonic stem cells (mESCs) using CHIR. Single DNase-seq sample in WT mesendodermal cells used as a reference

增强子功能的研究常采用截短报告基因实验或单基因缺失分析等碎片化手段，因此其功能受周边基因组环境影响的程度仍未明确。本研究依托靶向整合大片段DNA的Big-IN技术，解析了Igf2/H19基因座的调控架构——这是研究增强子选择性的经典模型。我们构建了携带157kb功能性Igf2/H19基因座的插入片段，并通过工程化突变在印记控制区（imprinting control region, ICR）定向调控CTCF结合，以此改变H19增强子簇的靶基因。将递送至内源基因座与安全港位点（Hprt）的插入片段的活性进行对比，结果显示，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基因座的组分与研究较为透彻的Sox2基因座进行互换后发现，H19增强子簇在脱离原生环境时活性显著降低，仅能在其原生周边环境中激活Sox2的表达。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，但其活性仅能被ICR处的CTCF结合状态部分调控。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中呈现紧密协同的调控模式，Sox2 LCR的调控活性则更为独立。我们证实，这类增强子簇可代表全基因组范围内更多基因座的调控特征。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；而我们所采用的合成调控基因组学方法，可实现完整基因座的大规模操控，以此探究基因座架构与其功能之间的关联。实验整体设计：采用CHIR试剂诱导小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）分化为工程化中内胚层细胞，对其进行脱氧核糖核酸酶I超敏位点测序（DNase-seq）图谱绘制；以野生型（WT）中内胚层细胞的单份DNase-seq样本作为对照参考