MOE Key Lab of Systems Biomedicine, Center for Comparative Biomedicine, State Key Lab of Oncogenes and Related Genes, Shanghai Cancer Institute, Joint International Research Laboratory of Metabolic & Developmental Sciences, Institute of Systems Biomedicine, Xin Hua Hospital, Shanghai Jiao Tong University, Shanghai, 200240, China.
The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China.
Genome Biol. 2020 Mar 23;21(1):75. doi: 10.1186/s13059-020-01984-7.
CTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem.
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.
These findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.
CTCF 是一种关键的绝缘子结合蛋白，哺乳动物基因组中包含大量的 CTCF 结合位点，其中许多位点串联排列。
我们使用 CRISPR DNA 片段编辑技术，结合染色体构象捕获技术，发现如果 CTCF 结合位点位于原钙黏蛋白（Pcdh）和β-球蛋白簇的增强子和启动子之间，它可以作为增强子阻断绝缘子，通过形成独特的定向染色质环，无论增强子是否含有 CTCF 结合位点。此外，计算机模拟、体内遗传缺失以及体外 dCas9 阻断实验表明，大量串联 CTCF 结合位点在 Pcdh 和免疫球蛋白重链（Igh）簇中可以实现细胞群体中平衡的启动子使用和单细胞中单等位基因表达的随机性。此外，CTCF 绝缘子通过与远端定向 CTCF 结合位点的长程染色质相互作用促进了，这与黏连蛋白的“环挤出”模型一致。最后，在全基因组范围内，基因表达水平与位于增强子和启动子之间的 CTCF 绝缘子呈负相关。因此，单个 CTCF 绝缘子确保了增强子的有效隔离和启动子的激活，而串联的 CTCF 拓扑绝缘子决定了平衡的空间接触和启动子选择。
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重要意义。
