Background CTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem. Results Using CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CTCF sites, if located between enhancers and promoters in the protocadherin ( Pcdh ) and β-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CTCF sites or not. Moreover, computational simulation in silico and genetic deletions in vivo as well as dCas9 blocking in vitro revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem CTCF sites in the Pcdh and immunoglobulin heavy chain ( Igh ) clusters. Furthermore, CTCF insulators promote, counter-intuitively, long-range chromatin interactions with distal directional CTCF sites, consistent with the cohesin “loop extrusion” model. Finally, gene expression levels are negatively correlated with CTCF insulators located between enhancers and promoters on a genome-wide scale. Thus, single CTCF insulators ensure proper enhancer insulation and promoter activation while tandem CTCF topological insulators determine balanced spatial contacts and promoter choice. Conclusions These findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.

中文翻译：

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串联 CTCF 位点作为绝缘体来平衡空间染色质接触和拓扑增强子 - 启动子选择

背景 CTCF 是一种关键的绝缘体结合蛋白，哺乳动物基因组包含许多 CTCF 位点，其中许多位点是串联​​组织的。结果使用 CRISPR DNA 片段编辑，结合染色体构象捕获，我们发现 CTCF 位点如果位于原钙粘蛋白 (Pcdh) 和 β-珠蛋白簇中的增强子和启动子之间，则通过形成不同的定向染色质环，无论增强子是否包含 CTCF 位点。此外，计算机模拟和体内遗传缺失以及体外 dCas9 阻断揭示了 Pcdh 和免疫球蛋白重链 (Igh) 簇中大量串联 CTCF 位点在细胞群中平衡的启动子使用和单细胞中的随机单等位基因表达. 此外，CTCF绝缘体促进，反直觉地，与远端定向 CTCF 位点的长程染色质相互作用，与黏连蛋白“环挤出”模型一致。最后，基因表达水平与位于全基因组范围内的增强子和启动子之间的 CTCF 绝缘子呈负相关。因此，单个 CTCF 绝缘体确保适当的增强子绝缘和启动子激活，而串联 CTCF 拓扑绝缘体确定平衡的空间接触和启动子选择。结论这些发现对拓扑染色质绝缘子在 3D 基因组折叠和发育基因调控中的作用具有有趣的意义。基因表达水平与位于全基因组范围内的增强子和启动子之间的 CTCF 绝缘子呈负相关。因此，单个 CTCF 绝缘体确保适当的增强子绝缘和启动子激活，而串联 CTCF 拓扑绝缘体确定平衡的空间接触和启动子选择。结论这些发现对拓扑染色质绝缘子在 3D 基因组折叠和发育基因调控中的作用具有有趣的意义。基因表达水平与位于全基因组范围内的增强子和启动子之间的 CTCF 绝缘子呈负相关。因此，单个 CTCF 绝缘体确保适当的增强子绝缘和启动子激活，而串联 CTCF 拓扑绝缘体确定平衡的空间接触和启动子选择。结论这些发现对拓扑染色质绝缘子在 3D 基因组折叠和发育基因调控中的作用具有有趣的意义。