High affinity enhancer promoter interactions can bypass strong CTCF/cohesin-mediated insulation contributing to phenotypic robustness [Capture Hi-C]. High affinity enhancer promoter interactions can bypass strong CTCF/cohesin-mediated insulation contributing to phenotypic robustness [Capture Hi-C]

Transcriptional control by enhancers located at large genomic distances from their targets is a common and integral feature of gene regulation. To understand how tissue specific enhancer-promoter interactions arise and to assess their resilience to perturbation of chromatin architecture, we generated an allelic series of mouse mutants carrying modifications to the structure of the Sox2 locus. We show that in pre-implantation epiblast cells and in neuronal lineages, CTCF-mediated loops are neither required for the interaction of Sox2 with its long-distance enhancers nor for its expression. Sox2-enhancer interactions in these cells were also robust to the introduction of CTCF/cohesin-mediated loops of varying degrees of insulation. These loops led to reduced interactions and Sox2 expression but did not cause their full disruption and were compatible with implantation and neurogenesis. In contrast, Sox2 expression in the anterior foregut was highly susceptible to perturbation of local chromatin structure with mutant embryos failing to separate trachea from esophagus and dying perinatally. Thus, in addition to being locus-specific, the functional impact of nuclear organization on cell fate-decisions is also highly dependent on biological context. Our work highlights the need of studying nuclear organization mechanisms in vivo and suggests that high-affinity enhancer-promoter interactions can provide robustness to structural perturbations to ensure faithful phenotypic outcomes. Overall design: Capture HiC (CHiC) was performed in the following tissues: blastocyst-derived mouse embryonic stem cells, heads of individual E11.5 embryos, sox2-expresssing anterior foregut cells. For ES and neural cells, 2 individual homozygous embryos were processed and anakyzed separately. WT controls are littermates from isolated embryos. AFG cells were pooled from a WT embryo.

位于距靶基因甚远的基因组位置的增强子所介导的转录调控，是基因调控中普遍且不可或缺的核心特征。为阐明组织特异性增强子-启动子相互作用的形成机制，并评估其对染色质结构扰动的耐受能力，我们构建了一系列携带Sox2基因座结构修饰的小鼠等位基因突变体。我们的研究发现，在植入前上胚层细胞与神经细胞谱系中，CCCTC结合因子(CTCF)介导的染色质环既非Sox2与其远端增强子相互作用所必需，也不影响其基因表达。在这类细胞中，Sox2与增强子的相互作用对引入不同程度绝缘功能的CTCF/黏连蛋白(cohesin)介导染色质环也表现出较强的耐受性。此类染色质环会降低Sox2与增强子的相互作用强度及其表达水平，但并未完全阻断二者的相互作用，且仍可支持胚胎植入与神经发生过程。与之相反，前肠前部组织中Sox2的表达对局部染色质结构的扰动极为敏感：突变胚胎无法将气管与食管分离，并于围产期死亡。由此可见，细胞核组织结构对细胞命运决定的功能性影响，不仅具有基因座特异性，还高度依赖于具体的生物学环境。本研究凸显了体内研究细胞核组织调控机制的必要性，并表明高亲和力的增强子-启动子相互作用可赋予染色质结构扰动耐受能力，从而保障表型结果的忠实性。实验整体设计：对以下组织开展捕获Hi-C(Capture HiC, CHiC)实验：囊胚来源的小鼠胚胎干细胞、单个胚胎发育第11.5天(E11.5)的胚胎头部、表达Sox2的前肠前部细胞。针对胚胎干细胞与神经细胞，我们分别处理并分析了2个独立的纯合子胚胎。野生型(WT)对照为分离自同窝的胚胎。前肠前部细胞取自1个野生型胚胎的混合细胞群。