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Cell | 上海市免疫学研究所叶菱秀团队与中科院分子细胞科学卓越创新中心孟飞龙团队合作揭示DNA柔性对抗体基因超突变谱式的塑造
发布日期:2023-04-25

科研进展                                                            
     

2023年4月24日,上海市免疫学研究所叶菱秀团队与中科院分子细胞卓越中心孟飞龙团队在Cell期刊在线发表了题为“Mesoscale DNA Feature in Antibody-Coding Sequence Facilitates Somatic Hypermutation”的研究论文。该论文从生化、细胞和小鼠模型三个水平全面揭示了抗体基因互补决定区(CDR)编码区偏好突变的分子基础,特别是抗体基因编码序列DNA柔性的重要生理作用,为下一代抗体基因人源化动物模型的设计奠定了理论基础。

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抗体可变区结构域由结合抗原的互补决定区(CDR)和维持免疫球蛋白结构的框架区(FR)组成。在体细胞高频突变过程中,胞苷脱氨酶AID偏好作用于WRC(W=A/T, R=A/G)基序,引入突变。其中,回文序列AGCT是AID最青睐的底物。然而,同样的AGCT却有不同的命运,往往表现出在CDR区域的高突变频率和在FR中的低突变频率。突变为什么具有偏好性最早于1982年由著名免疫学家David Baltimore与Klaus Rajewsky等提出,然而,近40多年来,造成“橘生淮南则为橘,生于淮北则为枳”这一现象的根本原因一直都没有令人非常信服的答案。

为了攻克这个长期困扰该领域的难题,研究团队从经典的生化实验出发,结合高通量测序技术,建立了体外检测抗体基因突变的新方法。研究发现生化实验可以重现体内CDR偏好突变特征。为了进一步确认CDR偏好突变是否具有进化保守性,研究人员将生化实验拓展至来自27个物种的1000余条抗体基因,发现这种偏好突变在很多物种中都普遍存在,尤其是在使用体细胞高频突变策略的四足动物(包括人、猴子、鼠、狗、羊驼和鸭嘴兽等)中高度保守(图1)。  

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图1  CDR偏好突变在多个物种中高度保守

CDR hypermutability is evolutionarily conserved  
随后,研究团队进行体内验证,聚焦于一段抗体基因CDR3序列,利用CRISPR-Cas9基因编辑技术对CDR3序列进行随机改造,快速获得了十几种不同序列环境的小鼠模型。通过对该区域突变热点AGCT突变频率的分析,发现序列改变影响了AGCT的突变频率,并且序列改变越靠近AGCT,对突变频率影响越大(图2  

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图2  CDR3区域DNA序列的改变影响突变频率
                    Mesoscale sequence determines the WRC mutability.  
为了进一步探寻DNA序列特征,研究人员利用分子动力学模拟、单分子生化方法,证明了DNA序列的柔性对AID的靶向过程发挥重要调控作用,尤其是WRC上游单链DNA序列的组成。结果显示,单链DNA的柔韧性与嘧啶-嘧啶二核苷酸的含量呈正相关,而抗体基因的CDR区域编码序列在进化中恰恰获得了这种高度柔性的特征(图3  

                    图片

图3                                         DNA序列柔韧性对突变频率的影响
Flexible DNA context promotes hypermutation
 

最后,研究人员在小鼠体内将一段柔性DNA序列插入低频突变区FR3,发现柔性序列极大地提高了FR3的突变频率,将FR3区逆转为类CDR区(图4

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图4  柔性序列将FR区逆转为类CDR区

Flexible DNA sequence makes a FR mutable.  
综上,该研究解答了长期困扰这一领域的难题,发现抗体基因CDR编码区DNA柔性促进了偏好性突变的发生;抗体基因编码序列具有调控AID突变靶向的非编码功能。这一工作为DNA力学性质参与调控细胞生命过程中提供了例证,也提示DNA柔性等力学性质可能在其他生命活动如肿瘤的发生发展中发挥重要作用。中尺度序列特征的发现,为设计下一代抗体基因人源化动物模型构筑了底层理论。  
中科院分子细胞科学卓越创新中心孟飞龙研究员和上海交通大学医学院上海市免疫学研究所叶菱秀研究员为该论文的共同通讯作者, 分子细胞科学卓越创新中心博士生王燕燕为该论文的第一作者。这项工作由王燕燕同学在上海市免疫学研究所从事研究助理工作时起始,然后在分子细胞中心攻读博士期间完成。该工作得到了上海交通大学达林泰教授、上海交通大学医学院郑小琪教授、哈佛大学医学院Frederick W. Alt教授、中国农大赵要风教授、瑞典卡罗林斯卡研究所Qiang Pan-Hammarström教授、中科院杭州医学所宋杰研究员、中科院生物物理所黄韶辉研究员、复旦大学曹志伟教授、分子细胞卓越中心刘珈泉研究员等合作实验室老师和同学的大力支持。该研究得到了国家重点研发计划、国家自然科学基金、上海市科技重大专项、上海市自然科学基金等资助。同时感谢上海交通大学医学院基础医学院和上海市免疫学研究所的公共技术平台以及免疫所与瑞金医院共建的免疫与疾病研究中心对本研究的大力支持。  


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叶菱秀,研究员,博士生导师。2010年博士毕业于剑桥大学, 2010~2015年于哈佛大学医学院进行博士后研究,2016年加入上海交通大医学院上海市免疫学研究所并担任抗体多样化课题组长。获得国家自然科学基金优秀青年项目、国际合作项目等基金资助。叶菱秀课题组长期致力于B淋巴细胞抗体多样化分子机制研究,特别是广谱中和抗体与自身抗体产生机制。课题组在体液免疫反应和中和抗体发现上积累了多种技术和完善的研究体系。实验室已培养“博新计划”博士后、博士研究生多名。欢迎对本课题组研究感兴趣的学生及博士后的加入,共同探讨推进B细胞抗体多样化研究的进展。             欢迎感兴趣的申请者来函咨询yeaplengsiew@shsmu.edu.cn
课题组主页为:
            https://www.shsmu.edu.cn/sii/info/1053/1455.htm                                        

 

 
Research Progress            

Study in Cell reveals the DNA flexibility feature in antibody gene sequence promotes somatic hypermutation


Researchers from the laboratory of Leng-Siew Yeap at the Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine and Fei-Long Meng at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences have reported the results of their groundbreaking study to elucidate the mechanisms that promote somatic hypermutation at the antigen-binding sites of antibody genes. These findings resolve a long-standing question that has puzzled antibody researchers for more than 40 years and provide new insights into the development of the next-generation humanized antibody animal models.

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One of the long-standing questions puzzling antibody researchers is why somatic hypermutations are concentrated in the three short, non-consecutive complementarity determining regions (CDRs) in the antibody gene sequence. First documented by the labs of David Baltimore, Klaus Rajewsky, and Leroy Hood in two Cell papers in the early 1980s, CDR hypermutation is well accepted and taken for granted by immunologists as an axiom in textbooks, although the mechanism is unknown. The study, published online on April 24, 2023 in Cell, reports that the flexible single-stranded DNA feature is the key to CDR-hypermutation. Using a cutting-edge, high-throughput biochemical assay that can test many DNA substrates for their deamination/mutation ability, the researchers found that the CDR hypermutation is evolutionarily conserved in species that use somatic hypermutation to diversify their antibody repertoire (Figure 1). Using the powerful passenger antibody gene allele mouse model system, which allows the detection of unselected mutational events, and the CRISPR/Cas9 system, which allows rapid generation of mice with DNA sequence alterations, the researchers found that the CDR hypermutability depends on the DNA sequence context at the mesoscale level (5-50 bp) (Figure 2). Using a combination of molecular dynamics simulations and single-molecule biochemistry, the researchers demonstrated that the targeting preference of the DNA mutator enzyme, activation induced cytidine deaminase (AID), is directly regulated by the flexibility of the single-stranded DNA substrate (Figure 3). Analysis of the antibody gene sequence showed that DNA sequences encoding the CDRs have evolved highly flexible properties to facilitate hypermutation, revealing a non-coding role of these sequences, and explaining the hypermutation pattern in lymphoma. Finally, regions that are normally “cold” for hypermutation can be made “hot” by engineering flexible DNA sequences in the “cold” regions (Figure 4), opening the door to the next generation of humanized animal models for antibody discovery.

     

The first author of this paper is Yanyan Wang, a research assistant who initiated this project and made the initial discoveries in mouse models in the Yeap lab, and continued to work on this exciting project as a Ph.D. student in the Meng lab. We would like to thank all of our wonderful collaborators on this study, Dr. Frederick Alt at Harvard Medical School, Dr. Qiang Pan-Hammarström at Karolinska Institute, Dr Lin-Tai Da at Shanghai Jiao Tong University, Dr. Xiaoqi Zheng at Shanghai Jiao Tong University School of Medicine, Dr. Yaofeng Zhao at China Agricultural University, Dr. Jie Song at Hangzhou Institute of Medicine, Dr. Shaohui Huang at University of Chinese Academy of Sciences, Dr. Zhiwei Cao at Fudan University and Dr Jiaquan Liu at Shanghai Institute of Biochemistry and Cell Biology. This work was supported by National Natural Science Foundation of China, National Key R&D Program of China, and etc. The authors also acknowledge the support of the Center of Immune-Related Diseases at Shanghai Institute of Immunology and Core Facilities at the Shanghai Institute of Immunology and the School of Basic Medical Science.


The Yeap lab welcomes enthusiastic students and postdoctoral fellows to join the group. Interested individuals can contact Dr Yeap at yeaplengsiew@shsmu.edu.cn

SII website: https://www.shsmu.edu.cn/sii/info/1164/2452.htm



Yanyan Wang’s poster presentation at SII-CIML symposia, 2018.

First from left: Dr Leng-Siew Yeap, second from right: Yanyan Wang.


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