Targeted DNA cleavage at switch regions in immunoglobulin class switch recombinat

描述（由申请方提供）：免疫球蛋白（IG）类别转换重组（CSR）是一种B细胞交换IG重链恒定结构域以实现病原体最佳清除的过程。这种独特的DNA重组是由DNA酶长转换区指导的，需要B细胞特异性因子激活诱导的胞苷脱氨酶（AID）以及其他普遍表达的DNA修复因子。众所周知，CSR是由AID催化的胞苷脱氨基作用引发的，导致开关区域中的尿嘧啶。然而，开关区指导AID顺式作用的机制以及最终导致DNA双链断裂的尿嘧啶修复途径的相互作用仍然不清楚。本申请的目的是鉴定开关区中的顺式作用DNA序列和在类别转换重组期间负责在限定的基因组基因座处靶向DNA切割的反式作用蛋白因子。我们已经开发了一种基于细胞的类别转换试验，用于研究内源性染色体位点的转换区序列的功能。该试验基于我们最近在CH 12 F3细胞中高效基因靶向的成功，CH 12 F3细胞是一种能够在体外产生鲁棒的奎宁诱导的CSR的小鼠B细胞系。我们设计了一种有效的敲入策略，以允许评估大量的开关区突变。在CH 12 F3细胞中的高效基因靶向还允许在CSR的细胞模型中通过反向遗传方法研究基因功能。我们现在能够解决几个重要但尚未回答的问题，以前很难在动物模型中解决。我们提出了三个具体的目标：（1）确定类转换重组所需的短序列基序;（2）确定类转换重组所需的长序列组织;（3）确定开关区域的DNA切割活性。该项目的完成将导致对开关区序列的功能的更完整的理解，并鉴定参与CSR中DNA切割的核酸酶。这些发现将为涉及类别转换重组的各种人类疾病提供机制见解。公共卫生相关性：这个项目的重点是阐明类别转换重组的分子机制，这是一个B细胞改变抗体同种型以最佳消除病原体的过程。理解类别转换重组的机制对于生物学原因（独特的区域特异性DNA重组）、免疫学原因（感染、过敏和自身免疫）及其癌症相关性（多种B细胞淋巴瘤中的致癌染色体易位）是重要的。

DESCRIPTION (provided by applicant): Immunoglobulin (Ig) class switch recombination (CSR) is a process by which B cells exchange the constant domain of the Ig heavy chain for the optimal clearance of pathogens. This unique DNA recombination is directed by kilobase- long switch regions and requires B cell-specific factor activation-induced cytidine deaminase (AID) as well as other ubiquitously expressed DNA repair factors. It is known that CSR is initiated by AID-catalyzed cytidine deamination resulting in uracils in the switch regions. However, the mechanism by which switch region directs AID actions in-cis and the interplays of uracil repair pathways that ultimately lead to DNA double strand breaks remain poorly defined. The objectives of this application are to identify cis-acting DNA sequences in the switch region and trans-acting protein factors that are responsible for targeted DNA cleavage at defined genomic loci during class switch recombination. We have developed a cell-based class switch assay for studying the function of switch region sequences at the endogenous chromosomal locus. This assay was based on our recent success in highly efficient gene targeting in CH12F3 cells, a mouse B cell line capable of robust cytokine-induced CSR in vitro. We have designed an efficient knock-in strategy to allow assessment of a large number of switch region mutations. Highly efficient gene targeting in CH12F3 cells also allows study of gene function by the reverse genetic approaches in a cellular model for CSR. We are now in position to address several important yet unanswered questions previously difficult to address in animal models. We propose three specific aims: (1) Identify short sequence motifs required for class switch recombination; (2) Identify long sequence organizations required for class switch recombination; (3) Identify the DNA cleavage activity at switch regions. The completion of this project will lead to a more complete understanding of the function of switch region sequences and the identification of the nucleases involved in DNA cleavage in CSR. These findings will provide mechanistic insight to a variety of human diseases involving class switch recombination. Public Health Relevance: This proposed project focuses on elucidating the molecular mechanism of class switch recombination, a process by which the B cell changes the isotype of the antibody for optimal elimination of pathogens. Understanding the mechanism of class switch recombination is important for biological reasons (a unique regional- specific DNA recombination), immunological reasons (infection, allergy and autoimmunity) and its cancer-relevance (oncogenic chromosomal translocations in a variety of B cell lymphomas).