Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [VII]. Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection [VII]

By CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation in silico and genetic deletions in vivo revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation. Overall design: To investigate the impacts of insertion or deletion of CTCF sites on 3D chromatin conformation and gene expression, chromasome conformation capture, ChIP-seq, and RNA-seq experiments were performed on DNA-fragment editting cell lines and transgenic mice.

本研究通过成簇规律间隔短回文重复序列（CRISPR）DNA片段编辑结合染色质构象捕获（chromosome conformation capture）技术，发现CTCF结合位点（CTCF binding site, CBS）若在成簇原钙粘蛋白（clustered Protocadherin, Pcdh）与β-珠蛋白基因簇中位于增强子与启动子之间，则可通过形成特定方向性染色质环发挥增强子阻断型绝缘子（enhancer-blocking insulator）的功能，且不受增强子自身是否包含CBS的影响。此外，硅基模拟（in silico）与体内基因敲除实验证实，大量串联可变CBS阵列可在细胞群体中维持启动子使用平衡，并在单细胞层面介导随机单等位基因表达。最终，全基因组范围内，位于增强子与启动子之间的CBS绝缘子与基因表达水平呈负相关关系。综上，单个CBS绝缘子可保障正确的增强子阻断与启动子激活，而串联排列的CBS绝缘子则决定启动子的选择性平衡。这一发现对拓扑绝缘子在三维基因组折叠与发育基因调控中的作用具有重要启示意义。整体实验设计：为探究CTCF位点的插入或缺失对三维染色质构象与基因表达的影响，本研究对DNA片段编辑细胞系及转基因小鼠开展了染色质构象捕获、染色质免疫共沉淀测序（ChIP-seq）与RNA测序（RNA-seq）实验。