VDJ Recombination Activating Genes-Functional Analysis

VDJ重组激活基因-功能分析

• 批准号: 6525667
• 负责人: MARJORIE A OETTINGER
• 金额: $ 38.69万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525667
• 关键词: DNA binding protein; DNA damage; T cell receptor; chemical cleavage; enzyme activity; gene deletion mutation; gene expression; gene induction /repression; gene rearrangement; genetically modified animals; immunoglobulin genes; immunoprecipitation; laboratory mouse; molecular cloning; molecular genetics; nucleic acid sequence; polymerase chain reaction; protein structure function; structural genes; tissue /cell culture; transfection /expression vector; yeast two hybrid system

A central process in the development of a competent immune system is the series of genomic rearrangement events that take place to produce a mature, functional immunoglobulin or T cell receptor gene. The failure of developing lymphocytes to correctly assemble their antigen receptor genes can lead to lymphoid tumors or immunodeficiency. As we previously demonstrated, the lymphoid-specific RAGI and RAG2 proteins carry out the first stage of this complex process, known as V (D) J recombination, by cleaving DNA at recombination signal sequences. V (D) J rearrangement is subject to many layers of regulation, from the simplest level of protein: DNA recognition between the RAG proteins and the signal sequences to the highly complex coordination of lineage- and stage-specific accessibility of the endogenous loci. Our goal over the next years is to understand the molecular basis for these different layers of regulation. First, we will address the requirements for assembling an active cleavage complex of RAG proteins with a pair of recombination signals on naked DNA templates. We will use a combination of genetic and biochemical approaches to ask how the RAG proteins recognize the RSS and how clevage of a signal pair is coordinated. We will also study a series of mutant derivatives of an accessory factor (mammalian HMG1 or yeast NHP6A) that is required for the assembly of a functional RAG cleavage complex, to understand this factor's role in V (D) J cleavage. Second, we will ask how chromatin structure and higher order DNA architecture can be modified to serve as a regulatable impediment to V (D) J cleavage. We will develop and analyze model substrates of increasing complexity that allow us to test what features of chromatin structure, what type of chromatin modifications and what chromatin modifying activities can serve to render a substrate more or less accessible to RAG-mediated cleavage. Third, we will use a series of genetically modified mice in which rearrangement is arrested at different stages of development to ask what chromatin modifications and modifying activities are present in vivo at endogenous antigen antigen receptor loci that, by their correlation with V (D) J cleavage, may be critical for regulating the reaction. In summary, the proposed experiments should yield a greater molecular understanding of the complex process of regulating V (D) J recombination during lymphoid development.

有能力的免疫系统发育的一个核心过程是一系列基因组重排事件，这些事件发生以产生成熟的、有功能的免疫球蛋白或T细胞受体基因。 发育中的淋巴细胞无法正确组装其抗原受体基因可能会导致淋巴肿瘤或免疫缺陷。 正如我们之前所证明的，淋巴特异性 RAGI 和 RAG2 蛋白通过在重组信号序列处切割 DNA 来执行这一复杂过程的第一阶段，即 V (D) J 重组。 V (D) J 重排受到多层调控，从最简单的蛋白质水平：RAG 蛋白质和信号序列之间的 DNA 识别，到内源基因座的谱系和阶段特异性可及性的高度复杂的协调。 我们未来几年的目标是了解这些不同调控层面的分子基础。首先，我们将解决在裸 DNA 模板上组装带有一对重组信号的 RAG 蛋白活性裂解复合物的要求。 我们将结合使用遗传和生化方法来探究 RAG 蛋白如何识别 RSS 以及如何协调信号对的切割。 我们还将研究组装功能性 RAG 裂解复合物所需的辅助因子（哺乳动物 HMG1 或酵母 NHP6A）的一系列突变衍生物，以了解该因子在 V (D) J 裂解中的作用。 其次，我们将询问如何修改染色质结构和高阶 DNA 结构以充当 V (D) J 裂解的可调节障碍。 我们将开发和分析日益复杂的模型底物，使我们能够测试染色质结构的哪些特征、染色质修饰的类型以及哪些染色质修饰活性可以使底物或多或少地易于 RAG 介导的切割。 第三，我们将使用一系列基因改造小鼠，其中重排在不同发育阶段被阻止，以探究体内内源性抗原抗原受体位点存在哪些染色质修饰和修饰活性，通过它们与 V (D) J 的相关性裂解，可能对于调节反应至关重要。 总之，所提出的实验应该对淋巴发育过程中调节 V (D) J 重组的复杂过程产生更深入的分子理解。