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一种可访问的或开放的免疫球蛋白MU(IgM)染色质结构。提高可获得性对于将静止的造血前体IgM基因座转换为具有生物功能的或激活的B血统第一个成员的基因座至关重要。然而，调节IgM可及性的机制尚不清楚。这项拟议的工作将集中于确定在B细胞发育过程中，一个不可接近的或封闭的MU染色质结构如何变得可接近，然后在成熟的B细胞中保持可接近。这项工作将回答这个问题：在染色质的背景下，MU增强子是如何激活的？完成拟议的工作代表着朝着确定驱动组织特异性IgM转录调控的机制这一长期目标迈出了第一步。来自多个研究人员长达20年的研究表明，染色质包装的变化调节B细胞发育过程中MU增强子的激活。MU增强子可及性增加，这是完全激活的前奏，与转录因子结合和包装MU增强子的组蛋白修饰有关。然而，显示转录因子引导染色质结构变化的直接“因果”关系尚未建立。总体而言，我们将通过三种方法检验这一假设，即Mu增强子因此B细胞的发育受转录因子指导的组蛋白修饰的调控：1.我们将通过在细胞中异地表达转录因子来定义从自然染色质区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Mu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导致u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u基因座可及性的细节，将通过阻断或加强这一发育关键过程，为药物控制疾病状态下B细胞的产生提供合理的靶点。此外，了解组织特异性基因表达的规则将使从癌症到糖尿病的各种单组织综合征的治疗能够得到精确的调控。

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.