利用B细胞抗体类别转换模型进行A-EJ修复通路的研究

发布时间：2018-04-11 15:07

  本文选题：非经典末端连接 + 抗体类别转换　； 参考：《北京协和医学院》2017年博士论文

【摘要】：保持基因组的完整性和稳定性对于生物体的存活是至关重要的。DNA双链断裂(DSB)是所有DNA损伤中最为严重的一种,可以通过外界因素(如电离辐射和化学诱变剂)和内源因素(如内源性DNA氧化以及DNA复制压力)产生。哺乳动物体内的DSB修复方式主要有两种:依赖于同源序列的同源重组(HR)和不依赖于同源序列的非同源末端连接(NHEJ)。近几年的研究发现,在经典的非同源末端连接(C-NHEJ)缺失的情况下,DSB末端仍然可以被有效地修复,预示着非经典末端连接(A-EJ)修复通路的存在。A-EJ活性已经在多种系统中得到证实,特别是在C-NHEJ核心因子(如DNA连接酶IV)缺陷的B细胞中,A-EJ介导的IgH基因座位置的抗体类别转换(CSR)效率是野生型细胞的50%。大量研究已经证实A-EJ的显著特点是对微同源序列(MH)的偏好性,以及在染色体转位过程中的重要作用。虽然目前已经有多个蛋白因子被报道参与A-EJ过程,但是其结果往往存在争议,特别是对参与A-EJ修复的DNA连接酶的研究还很不清楚。本研究利用CRISPR/Cas9系统,在Lig4缺陷的小鼠B细胞系CH12F3中完全敲除Lig1或者细胞核内的Lig3,建立只含有单个DNA连接酶(分别为Lig3和Lig1)的CH12F3细胞系。令人惊讶的是,只含有Lig1或Lig3的CH12F3细胞仍然具有A-EJ活性,可以有效地催化IgH基因座的CSR过程以及同一条染色体上由CRISPR/Cas9产生的DSB之间的染色体缺失;并且其活性与Lig1/Lig3同时存在的细胞没有明显差别。然而,在催化染色体转位方面,含有Lig1和Lig3的复合物显示出不同的特性。完全敲除细胞核中的Lig3,Lig4-/-细胞的染色体转位明显减少,而在完全敲除Lig1的细胞中则没有这种变化。另外,染色体转位连接处微同源序列的分析揭示了不同DNA连接酶催化活性的特异性。这些数据表明存在多种含DNA连接酶的复合物用以催化A-EJ过程。另外,本研究在小鼠B细胞中建立了 CRISPR/Cas9高通量筛选方法,在野生型和Lig4缺陷的细胞中筛选参与CSR过程中DSB修复的基因。首先建立了 Cas9稳定表达的CH12F3细胞系(野生型和Lig4缺陷型),并对sgRNA筛选的整个过程进行了优化。利用优化的实验条件,在Cas9稳定表达的WT和Lig4-/-CH12F3细胞中进行sgRNA文库的高通量筛选。对不同细胞群中的sgRNA进行高通量测序,通过MAGeCK分析方法,筛选IgA阳性细胞群中显著减少和IgA/IgM双阴性细胞群中显著增加的基因。在WT细胞中,筛选得到了 DNA-PKcs和Lig4等核心C-NHEJ蛋白因子,以及一些已知的参与细胞因子介导的CSR过程的基因。对其它未知功能的候选基因进行检测,发现Brd9和Fbxo28缺失严重影响细胞因子介导的CSR。目前正在对这一结果进行进一步验证。同时,基于Cas9/sgRNA介导的CSR模型的高通量筛选也正在进行。
[Abstract]:Maintaining the integrity and stability of the genome is essential to the survival of organisms. DSBs are the most serious of all DNA damage.It can be produced by external factors (such as ionizing radiation and chemical mutagens) and endogenous factors (such as endogenous DNA oxidation and DNA replication pressure).There are two main ways of DSB repair in mammals: homologous recombination of DSB which is dependent on the homologous sequence and non-homologous terminal ligation of DSB that does not depend on the homologous sequence.In recent years, it has been found that the end of DSB can still be repaired effectively in the absence of classical non-homologous terminal junctions (C-NHEJEJB), indicating that the existence of the repair pathway of non-classical terminal junctions (EJA-EJA-EJB) has been confirmed in many systems.Especially in B cells with C-NHEJ core factors (such as DNA ligase IV-deficient), the efficiency of A-EJ mediated antibody class conversion of IgH locus is 50% of wild-type cells.A large number of studies have confirmed that A-EJ is characterized by its preference for microhomologous sequences and its important role in chromosome translocation.Although several protein factors have been reported to be involved in the A-EJ process, the results are often controversial, especially the study of the DNA ligase involved in A-EJ repair is still unclear.In this study, CRISPR/Cas9 system was used to completely knockout Lig1 or Lig3 in Lig4 deficient mouse B cell line CH12F3, and to establish CH12F3 cell line containing only a single DNA ligase (Lig3 and Lig1, respectively).Surprisingly, CH12F3 cells containing only Lig1 or Lig3 still have A-EJ activity, which can effectively catalyze the CSR process of IgH locus and the deletion of DSB between DSB produced by CRISPR/Cas9 on the same chromosome.And its activity is not significantly different from that of Lig1/Lig3 cells.However, complexes containing Lig1 and Lig3 exhibit different characteristics in catalytic chromosomal translocation.The chromosomal translocation of Lig3 + Lig4-r- cells in the complete knockout nucleus was significantly reduced, but there was no such change in the cells with complete knockout of Lig1.In addition, the specific catalytic activity of different DNA ligases was revealed by the analysis of the microhomologous sequences at the chromosomal translocation junctions.These data indicate that there are many complexes containing DNA ligase to catalyze the A-EJ process.In addition, a high-throughput CRISPR/Cas9 screening method was established in mouse B cells to screen genes involved in DSB repair in wild type and Lig4 deficient cells.Firstly, CH12F3 cell lines (wild type and Lig4 deficient type) expressing Cas9 stably were established, and the whole process of sgRNA screening was optimized.High throughput screening of sgRNA library was carried out in WT and Lig4-/-CH12F3 cells stably expressed by Cas9 using optimized experimental conditions.The high throughput sequencing of sgRNA in different cell groups was carried out. The genes in IgA positive cell group and IgA/IgM double negative cell group were significantly decreased and significantly increased by MAGeCK analysis.In WT cells, core C-NHEJ protein factors such as DNA-PKcs and Lig4, as well as some known genes involved in the cytokine mediated CSR process, were screened.Candidate genes of other unknown functions were detected and the deletion of Brd9 and Fbxo28 seriously affected cytokine mediated CSRs.This result is currently being further validated.At the same time, high throughput screening of CSR model based on Cas9/sgRNA is also underway.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.5

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