STRUCTURE/FUNCTION OF CHROMATIN IN B-CELL DEVELOPMENT

B 细胞发育中染色质的结构/功能

• 批准号: 2189492
• 负责人: VERONICA C BLASQUEZ
• 金额: $ 9.53万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2189492
• 关键词: B lymphocyte; DNA binding protein; DNA footprinting; animal genetic material tag; cell differentiation; chemical structure function; chromatin; cytokine; genetic enhancer element; genetic transcription; immunoglobulin genes; laboratory mouse; mitogens; tissue /cell culture

The B-cell developmental system is a paradigm for understanding the mechanisms that enable an organism to respond to extracellular signals. Resting B-cells respond to antigenic stimuli by secretion of immunoglobulin molecules. One of the hallmarks of this terminal differentiation events is an increase in the expression of immunoglobulin molecules reflected in the increased levels of mRNA. Although a large number of studies have been exerted in elucidating the molecular basis for this alteration in the level of gene expression, we still know very little about the mechanisms involved in this process. Our studies of the mouse kappa immunoglobulin locus has revealed that the structure of chromatin around the 3' enchancer region is remarkably different in antibody-secreting plasma B-cells compared with that of mature B-cells. The DNAse I hypersensitive structure first observed in pro B-cells persists at the mature B-cell stage but undergoes a transformation at some stage between the differentiation of B-cells to plasma cells resulting in the spreading of nuclease susceptible sites upstream and downstream of the initial site. Since similar changes are detected in terminally differentiated plasma cells expressing lamba chains, these results suggest that the alteration in the 3'region is a differentiation- dependent event. These changes in chromatin structure may underlie an important aspect of B-cell end-differentiation an might bear upon kappa gene expression. By a combination of reverse genetic, immunological, cell biological, molecular biological, and biochemical techniques, we hope to identify the molecular basis of these chromatin structural changes. We have the unique opportunity to identify the factors that participate in terminal differentiative events in B-cell development, the external agents that these factors respond to, and their possible mechanism of action on gene expression. Specifically, we wish to address the following specific aims: 1) To determine the effects of extracellular stimuli (mitogens and cytokines) on the chromosomal alterations observed during B-cell maturation. This should lead to the establishment of an in vitro system for studying the mechanisms involved in 3' enhancer induction. 2) To localize the DNA sequences within the 3" enhancer that define the formation of DNAse I hypersensitive sites. 3) To identify the precise nucleotide sequences in contact with protein factors in vivo genomic footprinting. 4) To identify and biochemically characterize the protein factors that bind the DNA sequences identified in specific aim#2.

B细胞发育系统是理解的范式 使生物体能够对细胞外信号做出反应的机制。 静止的B细胞通过分泌对抗原刺激做出反应 免疫球蛋白分子。 这个航站楼的标志之一 分化事件是免疫球蛋白表达的增加 分子反映在mRNA水平升高中。 虽然很大 研究的数量已在阐明分子基础上施加 对于基因表达水平的这种改变，我们仍然非常了解 关于此过程中涉及的机制几乎没有。 我们对 小鼠Kappa免疫球蛋白基因座表明 3'辅助器区域周围的染色质在 与成熟的B细胞相比，抗体分泌血浆B细胞。 DNase I超敏结构首先在Pro B细胞中观察到 在成熟的B细胞阶段持续存在，但在 B细胞与浆细胞的分化之间的某个阶段 导致核酸酶在上游易感位点的扩散和 初始站点的下游。 由于在 终端分化的表达lamba链的浆细胞，这些 结果表明，3'Rigion的改变是一个分化 依赖事件。 染色质结构的这些变化可能是 B细胞终端分化的重要方面可能对Kappa承担 基因表达。 通过反向遗传，免疫学的结合 细胞生物学，分子生物学和生化技术，我们 希望识别这些染色质结构的分子基础 更改。 我们有独特的机会来确定因素 参加B细胞开发中的终端区分事件， 这些因素响应的外部代理及其可能 基因表达的作用机理。 具体来说，我们希望解决 以下具体目的： 1）确定细胞外刺激的影响 细胞因子）在B细胞期间观察到的染色体改变 成熟。 这应该导致建立体外系统 用于研究3'增强子诱导的机制。 2）将DNA序列定位在3“增强子中） DNase I超敏部位的形成。 3）确定与蛋白质接触的精确核苷酸序列 体内基因组足迹的因素。 4）识别和生化表征蛋白质因子 结合特定目标＃2中鉴定的DNA序列。