Targeted DNA cleavage at switch regions in immunoglobulin class switch recombinat

描述（由申请人提供）：免疫球蛋白（Ig）类开关重组（CSR）是B细胞交换Ig重链恒定结构域以最佳清除病原体的过程。这种独特的DNA重组是由千碱基长开关区引导的，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催化胞苷脱氨引起的开关区域尿嘧啶引发的。然而，开关区如何引导AID的顺式作用以及尿嘧啶修复途径的相互作用最终导致DNA双链断裂的机制仍不清楚。本应用程序的目的是鉴定开关区域中的顺式作用DNA序列和反式作用蛋白质因子，这些蛋白质因子在类开关重组期间负责在定义的基因组位点上的靶向DNA切割。我们开发了一种基于细胞的开关实验，用于研究内源性染色体位点开关区域序列的功能。该试验基于我们最近在CH12F3细胞中高效基因靶向的成功，CH12F3细胞是一种在体外能够强大的细胞因子诱导CSR的小鼠B细胞系。我们设计了一种有效的敲入策略来评估大量的开关区突变。CH12F3细胞中高效的基因靶向也允许在CSR细胞模型中通过反向遗传方法研究基因功能。我们现在可以解决以前在动物模型中难以解决的几个重要但未解决的问题。我们提出了三个具体目标：(1)识别类切换重组所需的短序列基序；(2)确定类开关重组所需的长序列组织；(3)鉴定开关区的DNA切割活性。本项目的完成将使我们对开关区序列的功能有更全面的了解，并对CSR中参与DNA切割的核酸酶进行鉴定。这些发现将为涉及类开关重组的各种人类疾病提供机制见解。公共卫生相关性：本项目旨在阐明类开关重组的分子机制，这是B细胞改变抗体同型以最佳消除病原体的过程。了解类开关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（各种B细胞淋巴瘤的致癌染色体易位）非常重要。

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).