Somatic Mutation of Ig Variable Regions

Ig 可变区的体细胞突变

• 批准号: 6775472
• 负责人: MATTHEW D SCHARFF
• 金额: $ 38.76万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6775472
• 关键词: B lymphocyte; acetylation; acyltransferase; aminohydrolases; antibody formation; cell line; chromatin; chromatin immunoprecipitation; cytidine; enzyme activity; enzyme mechanism; gene expression; gene mutation; gene targeting; genetic regulatory element; genetically modified animals; histones; immunoglobulin genes; laboratory mouse; nucleic acid structure; polymerase chain reaction

DESCRIPTION (provided by applicant): The goal of this proposal is to identify the molecular mechanisms responsible for targeting somatic hypermutation (SHM) to antibody variable regions. We and others have shown that activation induced cytidine deaminase (AID) is probably the only B cell specific protein that is required for SHM, but the mechanisms that target AID and the other ubiquitous factors involved in SHM to cytidines in hot spots in the V region have not been identified. We will determine if other macromolecules are associated with AID or are required to activate its enzymatic activity and examine their role in targeting AID to the heavy chain V region. In the previous grant period we have shown that this targeting is associated with the hyperacetylation of the histones associated with the V region but not with the C region that is protected from mutation. We propose to extend these findings by identifying any other chromatin modifications that occur and by determining the role of these modifications in remodeling chromatin to make the V region accessible to mutating factors or in recruiting the mutational factors to the V region. We will also identify the histone acetylases and deacetylases that carry out these modifications and use RNAi or gene replacement to determine if these enzymes are required for targeting of SHM to hot spots and to the V region. We will use gene replacement of mutated and deleted regulatory and other DNA elements to identify the cis-acting sequences in the Ig gene that play a role in the targeting of mutation either through the specific recruitment of the mutational factors or by mediating the changes in chromatin structure. Most of these experiments will be carried out Burkitt's lymphoma cells lines but we will confirm the major findings with normal B cells.

描述（由申请人提供）：该提案的目的是确定负责靶向躯体超突变（SHM）到抗体变量区域的分子机制。我们和其他人已经表明，激活诱导的胞苷脱氨酶（AID）可能是SHM所需的唯一B细胞特异性蛋白，但是尚未鉴定出靶向AID和其他无处不在的SHM涉及的SHM涉及的无处不在的因素到V区域中热点的细胞衍生物。我们将确定其他大分子是否与AID相关，或者需要激活其酶活性并检查其在靶向重链V区域中的作用。在上一个赠款期间，我们已经表明，该靶向与与V区域相关的组蛋白的高乙酰化有关，而与受保护不受突变的C区域无关。我们建议通过鉴定发生的任何其他染色质修饰，并确定这些修饰在重塑染色质中的作用，以使V区域可接近突变因子或将突变因子募集到V区域中，以扩展这些发现。我们还将确定进行这些修饰的组蛋白乙酰基酶和脱乙酰基酶，并使用RNAi或基因置换来确定是否需要将SHM靶向热点和V区域。我们将使用突变和删除的调节和其他DNA元件的基因替代基因来鉴定Ig基因中的顺式作用序列，这些序列通过突变因子的特定募集或介导染色质结构的变化来在突变靶向突变中起作用。这些实验中的大多数将进行伯基特的淋巴瘤细胞系线，但我们将确认正常B细胞​​的主要发现。