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重链的恒定结构域以最佳清除病原体的过程。这种独特的DNA重组是由千碱基长的切换区控制的，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催化的胞苷脱氨反应启动的，导致开关区域的尿嘧啶。然而，开关区域在顺式病毒中指导AID作用的机制以及尿嘧啶修复途径之间的相互作用最终导致DNA双链断裂的机制仍不清楚。这项应用的目的是确定在类开关重组过程中，顺式作用的DNA序列和反式作用的蛋白因子，这些顺式作用的DNA序列和反式作用的蛋白因子负责在特定的基因组座位上进行靶向DNA切割。我们建立了一种基于细胞的类开关试验，用于研究内源染色体座位上开关区域序列的功能。这项分析是基于我们最近在CH12F3细胞中高效基因打靶的成功，CH12F3细胞是一种能够在体外由细胞因子诱导CSR的小鼠B细胞系。我们设计了一种有效的敲入策略，可以评估大量的开关区域突变。CH12F3细胞中高效的基因打靶也允许在CSR的细胞模型中通过反向遗传方法研究基因功能。我们现在可以解决以前在动物模型中很难解决的几个重要但尚未回答的问题。我们提出了三个具体的目标：(1)确定类开关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)鉴定开关区域的DNA切割活性。该项目的完成将使人们对切换区序列的功能有更全面的了解，并对参与DNA切割的核酸酶进行鉴定。这些发现将为涉及类转换重组的各种人类疾病提供机械性的洞察。公共卫生相关性：这项拟议的项目侧重于阐明类转换重组的分子机制，即B细胞通过改变抗体的同种类型来最佳消除病原体的过程。了解类转换重组的机制对于生物学原因(一种独特的区域特异性DNA重组)、免疫学原因(感染、过敏和自身免疫)及其与癌症的相关性(各种B细胞淋巴瘤中的致癌染色体易位)都是重要的。

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).