Disruptions of Topological Chromatin Domains Causes Pathogenic Rewiring of Gene-Enhancer Interactions [RNA-Seq]. Mus musculus

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs) that have been proposed to represent large regulatory units. Here we demonstrate that disruption of TADs can cause rewiring of long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome. Overall design: RNA-seq profile of developing distal limbs of mutants and WT animals at E11.5哺乳动物基因组被组织为兆碱基级别的拓扑关联结构域（topologically associated domains, TADs），这类结构被认为是大型基因调控单元。本研究证实，TAD的破坏可引发远程调控架构的重连，并最终导致致病表型。我们发现，多种人类肢体畸形均由改变跨越TAD的WNT6/IHH/EPHA4/PAX3基因座结构的缺失、倒位或重复变异所导致。本研究借助CRISPR/Cas基因组编辑技术，构建了携带相应染色体重排的小鼠模型。无论是在小鼠肢体组织还是患者来源的成纤维细胞中，与疾病相关的结构变异均会引发启动子与非编码DNA之间的异位互作；同时，原本与Epha4相关的肢体增强子簇相对于TAD边界发生错位，进而驱动该基因座内另一基因出现异位肢体表达。本研究结果证实了TADs通过基因组架构调控基因表达的功能重要性，并为预测人类结构变异（尤其是人类基因组非编码区域的结构变异）的致病性提供了判定标准。实验整体设计：对E11.5发育阶段的突变体与野生型（WT）动物的远端肢体进行RNA测序（RNA-seq）谱分析。