串联反向CTCF位点的系列删除揭示增强子调控HOXD基因簇表达的平衡
收稿日期: 2021-04-11
修回日期: 2021-05-12
网络出版日期: 2021-06-30
基金资助
国家自然科学基金项目编号(31800636);国家自然科学基金项目编号(31630039);国家自然科学基金项目编号(91940303);上海市科学技术委员会项目资助编号(19JC1412500)
Serial deletions of tandem reverse CTCF sites reveal balanced HOXD regulatory landscape of enhancers
Received date: 2021-04-11
Revised date: 2021-05-12
Online published: 2021-06-30
Supported by
Supported by the National Natural Science Foundation of China Nos(31800636);Supported by the National Natural Science Foundation of China Nos(31630039);Supported by the National Natural Science Foundation of China Nos(91940303);Science and Technology Commission of Shanghai Municipality No(19JC1412500)
三维基因组染色质架构蛋白CTCF (CCCTC-binding factor)能够介导增强子与基因启动子的远距离染色质相互作用,也可以结合调控区域的绝缘子发挥增强子绝缘功能,对发育中的基因表达调控具有重要作用。同源框基因家族(Homeobox gene family, Hox)编码一类控制动物发育的关键转录因子,在发育中主要沿胚胎首尾轴(head-to-tail axis)呈时空线性表达。在哺乳动物中,Hox基因分为HoxA、HoxB、HoxC和HoxD四个基因簇,在中枢神经系统、骨骼和四肢发育中发挥重要功能。HoxD基因簇主要调控四肢发育,受位于其两侧调控域内的增强子调节,沿肢体近远轴(proximal-distal axis)呈时空线性表达。在人类基因组中,HOXD基因簇及其两侧的调控区域分布有串联排列的CTCF结合位点(简称CTCF位点),参与9个HOXD基因的表达调控。本研究以HOXD基因簇为模式基因,探究CTCF对发育基因(developmental genes)转录调控的影响。利用CRISPR DNA片段编辑技术在人HEK293T细胞中获得一系列的串联反向CTCF位点删除的单细胞克隆株。RNA-seq实验揭示CTCF位点删除后HOXD基因表达下降。定量高分辨率染色体构象捕获实验显示,HOXD与上游增强子簇的远距离染色质相互作用增强,与下游增强子簇的远距离染色质相互作用减弱。综上所述,串联反向的CTCF位点通过其绝缘子功能维持上下游增强子簇对HOXD基因簇表达调控的平衡,为探究动物发育过程中Hox基因表达的精准调控机制提供参考。
王玲, 李金环, 黄海燕, 吴强 . 串联反向CTCF位点的系列删除揭示增强子调控HOXD基因簇表达的平衡[J]. 遗传, 2021 , 43(8) : 775 -791 . DOI: 10.16288/j.yczz.21-132
The genome architectural protein CTCF (CCCTC-binding factor) not only mediates long-distance chromatin interactions between distal enhancers and target promoters, but also functions as an important insulator-binding factor to block improper enhancer activation of non-target promoters, and is thus of great significance to transcriptional regulation of developmental genes. The Hox (Homeobox) gene family plays an important role in the development of the brain, bones, and limbs. The spatiotemporal colinear expression of the HOXD cluster along the proximal-distal axis of limbs is regulated by two clusters of enhancers known as super-enhancers located in the flanking regulatory regions. We focused on the HOXD cluster to explore the architectural role of CTCF in transcriptional regulation of developmental genes. The HOXD cluster contains 9 paralogous genes intermixed with a series of CBS (CTCF-binding site) elements. Using the CRISPR DNA-fragment editing system, we generated a series of single-cell HEK293T clones with deletion of increasing numbers of reverse CBS elements. RNA-seq experiments revealed decreased levels of HOXD gene expression. In addition, chromosome conformation capture experiments revealed increased long-distance chromatin interactions between HOXD and the upstream enhancer cluster and corresponding decreased interactions between HOXD and the downstream enhancer cluster. Thus, tandem reverse CTCF sites function as insulators to maintain HOXD regulatory balance between the upstream and downstream enhancer clusters. This study has interesting implications on the precise gene expression control of the Hox family during animal development.
Key words： HOXD gene cluster; CTCF site; balanced regulatory mechanism; enhancer; insulator
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