Mechanisms regulating immunoglobulin gene transcription

描述（由申请人提供）：成功开发B细胞的第一步之一是建立可接近或开放的免疫球蛋白mu（IgM）染色质结构。增加的可及性对于将造血前体的静止IgM基因座转化为B谱系的第一定型成员中的生物学功能性或活化的基因座是关键的。然而，调节IgM可及性的机制尚不清楚。拟议的工作将集中在确定如何访问一个不可访问的，或关闭，μ染色质结构变得可访问的B细胞发育过程中，然后保持在成熟的B细胞。这项工作将回答这个问题：如何在染色质的背景下激活mu增强子？完成拟议的工作是朝着表征驱动IgM转录的组织特异性调节机制的长期目标迈出的第一步。来自多个研究者20年的工作表明，染色质包装的改变调节B细胞发育过程中mu增强子的激活。增加的mu增强子可及性，完全激活的前奏，与转录因子结合和修饰包装mu增强子的组蛋白相关。然而，一个直接的“因果”关系，显示转录因子指导染色质结构的变化尚未建立。总的来说，我们将通过三种方法来检验mu增强子以及因此B细胞发育受转录因子指导的组蛋白修饰调控的假设：1.我们将定义通过在细胞中异位表达转录因子来从天然染色质化的μ基因座建立最大可及性所需的蛋白质组合; 2.我们将破坏可及性因子的DNA结合活性，然后测量开放的μ染色质结构的稳定性; 3.我们将通过染色质免疫沉淀来测试诱导mu可及性的转录因子如何影响包装mu增强子的组蛋白的乙酰化和甲基化。了解B细胞发育过程中μ基因座可及性的细节将为通过阻断或增强这种发育关键过程来药物控制疾病状态下的B细胞生成提供逻辑靶点。此外，了解组织特异性基因表达的规则将允许对从癌症到糖尿病的各种单一组织综合征进行精确调节的治疗。

DESCRIPTION (provided by applicant): One of the first steps in successful B cell development is establishing an accessible, or open, immunoglobulin mu (IgM) chromatin structure. Increased accessibility is critical for converting the quiescent IgM locus of the hematopoietic precursor into a biologically functional, or activated, locus in the first committed member of the B lineage. However, the mechanisms regulating IgM accessibility are unknown. The proposed work will focus on determining how an inaccessible, or closed, mu chromatin structure becomes accessible during B cell development then remains accessible in the mature B cell. This work will answer the question: how is the mu enhancer activated in the context of chromatin? Completing the proposed work represents a first step towards the long-term goal of characterizing mechanisms driving tissue-specific regulation of IgM transcription. Work from multiple investigators spanning 20 years suggests that alterations in chromatin packaging regulate mu enhancer activation during B cell development. Increased mu enhancer accessibility, a prelude to full activation, correlates with transcription factor binding and modification of histone proteins packaging the mu enhancer. However, a direct "cause and effect" relationship showing transcription factors directing changes in chromatin structure has not been established. Overall, we will test the hypothesis that the mu enhancer, and hence B cell development is regulated by transcription factor-directed histone modifications through three approaches: 1. We will define the combination of proteins required for establishing maximal accessibility from a naturally chromatinized mu locus by ectopically expressing transcription factors in cells; 2. We will destroy DNA binding activity of the accessibility factors then measure stability of the open mu chromatin structure; 3. We will test how transcription factors that induce mu accessibility affect acetylation and methylation of the histones packaging the mu enhancer by chromatin immunoprecipitation. Understanding the details of mu locus accessibility during B cell development will provide logical targets for pharmaceutically controlling B cell generation in disease states by either blocking or enhancing this developmentally critical process. In addition understanding the rules for tissue-specific gene expression will allow exquisitely regulated delivery of treatments for a variety of single tissue syndromes from cancer to diabetes.