Mechanisms regulating immunoglobulin gene transcription

描述（由申请人提供）：成功的B细胞发育的第一步是建立一个可接近的或开放的免疫球蛋白（IgM）染色质结构。增加可及性对于将造血前体的静止IgM位点转化为具有生物学功能或激活的B谱系第一个成员的位点至关重要。然而，调控IgM可及性的机制尚不清楚。这项工作将侧重于确定在B细胞发育过程中不可接近或封闭的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：mu增强子是如何在染色质的背景下被激活的？完成拟议的工作是迈向描述驱动IgM转录的组织特异性调节机制的长期目标的第一步。20年来，多名研究人员的工作表明，染色质包装的改变调节了B细胞发育过程中mu增强子的激活。mu增强子可及性的增加是完全激活的前奏，与转录因子结合和修饰包装mu增强子的组蛋白有关。然而，转录因子指导染色质结构变化的直接“因果”关系尚未建立。总的来说，我们将通过三种方法验证mu增强子和B细胞发育受转录因子定向组蛋白修饰调节的假设：1。我们将通过在细胞中异位表达转录因子来确定从自然染色化的mu位点建立最大可及性所需的蛋白质组合；2. 我们将破坏可接近因子的DNA结合活性，然后测量开放mu染色质结构的稳定性；3. 我们将通过染色质免疫沉淀测试诱导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mu位点可及性的细节，将为在疾病状态下通过阻断或增强这一发育关键过程来药物控制B细胞的产生提供逻辑靶点。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.