遗传 ›› 2022, Vol. 44 ›› Issue (9): 783-797.doi: 10.16288/j.yczz.22-201

• 研究报告 • 上一篇    下一篇

靶向敲除β-珠蛋白基因座控制区增强子HS2对K562细胞转录组的影响

陈秀丽(), 黄海燕(), 吴强()   

  1. 上海交通大学系统生物医学研究院比较生物医学研究中心,系统生物医学教育部重点实验室,上海 200240
  • 收稿日期:2022-06-16 修回日期:2022-07-20 出版日期:2022-09-20 发布日期:2022-08-01
  • 通讯作者: 黄海燕,吴强 E-mail:chenxiuli@sjtu.edu.cn;hy_huang@sjtu.edu.cn;qwu123@gmail.com
  • 作者简介:陈秀丽,硕士研究生,专业方向:生物学。E-mail: chenxiuli@sjtu.edu.cn
  • 基金资助:
    国家自然科学基金项目(81872944);国家自然科学基金项目(31630039);上海市科学技术委员会项目资助(19JC1412500);上海市科学技术委员会项目资助(21DZ2210200)

Targeted deletion of 5′HS2 enhancer of β-globin locus control region in K562 cells

Xiuli Chen(), Haiyan Huang(), Qiang Wu()   

  1. Center for Comparative Biomedicine, Key laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China
  • Received:2022-06-16 Revised:2022-07-20 Online:2022-09-20 Published:2022-08-01
  • Contact: Huang Haiyan,Wu Qiang E-mail:chenxiuli@sjtu.edu.cn;hy_huang@sjtu.edu.cn;qwu123@gmail.com
  • Supported by:
    Supported by the National Natural Science Foundation of China(81872944);Supported by the National Natural Science Foundation of China(31630039);Science and Technology Commission of Shanghai Municipality Program Nos(19JC1412500);Science and Technology Commission of Shanghai Municipality Program Nos(21DZ2210200)

摘要:

人类β-地中海贫血的发病机制与β-样珠蛋白基因异常表达息息相关。人类β-样珠蛋白基因以5′-ε-Gγ-Aγ-δ-β-3′的顺序排列于β-珠蛋白基因座,受5′LCR (locus control region)中5个超敏位点(hypersensitive site, HS)5′HS5~5′HS13′HS1调控。其中5′HS2是最重要的增强子,能产生增强子RNA (enhancer RNA)并调控ε-globin、γ-globin和β-globin的表达。为了进一步探究K562细胞中增强子5′HS2的功能,本研究首先通过染色质构象捕获技术在人慢性髓原白血病K562细胞中探测到5′HS2介导的染色质相互作用集中在以包含CTCF (CCCTC-binding factor)位点的3′HS1和5′HS5为边界的拓扑结构域中,5′HS2在三维空间上与HBE1、HBG2HBG1启动子区域相互靠近。其次运用CRISPR DNA片段编辑技术在K562细胞系中删除了增强子5′HS2。最后通过RNA-seq和CUT&Tag (cleavage under target & tagmentation)实验分析两个5′HS2删除的单克隆细胞系的转录组和染色质H3K27ac组蛋白修饰,发现91个基因表达显著下调而且其启动子区的H3K27ac修饰程度显著降低。这些基因主要聚类于氧气运输、免疫应答、细胞粘附、抗氧化和维持血栓形成等与红细胞功能相关的生物过程中,表明K562细胞系中增强子5′HS2对红细胞功能相关基因的转录产生了广泛影响。

关键词: β-珠蛋白基因座, 增强子, 5′HS2, 三维基因组, H3K27ac

Abstract:

Human β-thalassemia is closely associated with aberrant expression of β-like globin genes. Human β-like globin genes are organized in the order of 5′-ε-Gγ-Aγ-δ-β-3′ within the β-globin locus. The expression of β-like globin genes is regulated by 3′HS1 and five DNase I hypersensitive sites (5′HS5~5′HS1) in a locus control region. The 5′HS2 enhancer transcribes enhancer RNA and regulates the expression of ε-globin, γ-globin and β-globin. To further study the function of 5′HS2, we detected the local 3D genomic architecture via chromatin conformation capture experiments and used CRISPR/ Cas9-based DNA fragment editing to delete 5′HS2 in human K562 leukaemia cells. In this study, we found that 5′HS2-mediated chromatin interactions were enriched in a topologically associated domain that was bordered by 3′HS1 and 5′HS5. Within this topologically associated domain, 5′HS2 is highly close to the promoter regions of HBE1, HBG2 and HBG1. Upon deletion of the 5′HS2 enhancer, 91 genes were significantly down-regulated with reduced abundance of H3K27ac at their promoter regions. These down-regulated genes are mainly associated with oxygen transport, immune response, cell adhesion, anti-oxidant and thrombosis. These data suggested that many genes associated with functions of erythrocytes were decreased at transcriptional levels upon deletion of the 5′HS2 enhancer.

Key words: β-globin locus, enhancer, 5′HS2, 3D genome, H3K27ac