REGULATION OF IMMUNOGLOBULIN GENE EXPRESSION IN B CELLS

免疫球蛋白μ重链基因增强子激活分化中的B淋巴细胞中的转录和重组。增强子活性由几种DNA结合蛋白介导，这些蛋白在增强子上组装成精确的多蛋白复合物。 该计划的长期目标是了解增强子功能的分子机制。在本申请中将解决增强子功能的两个方面。 首先，将检查组合转录控制的机制（具体目标1）。组合控制是指增强子的中心特征，由此增强子的性质与与增强子相互作用的单个DNA结合蛋白的性质非常不同。 生化和遗传实验，建议了解增强子上的远端位点合作激活转录和重组的机制。其次，将在体外（特异性目标2）和B淋巴细胞（特异性目标3）中研究增强子调节染色质结构的机制。 体外染色质组装将用于检查mu增强子结合蛋白的单独和组合的作用。 将通过DNAase I和微球菌核酸酶消化试验评估核小体定位和染色质结构的结构后果。 与此同时，将通过体外转录和重组试验评价染色质重构的功能后果。 最后，前B细胞中未重排IgH基因座的染色质结构将通过DNAase I、微球菌核酸酶和组蛋白乙酰化来绘制。 将定义基因座可及性的限度，并通过分析序列已被删除的细胞来确定重要调控元件的贡献。 总之，这些研究将为mu增强子的功能提供全面的见解。深入了解组织特异性基因调控将是必不可少的方法，以选择性地减少异常基因表达的疾病状态，反之，以提高基因表达，以克服免疫缺陷。 组合转录激活的规则也将允许设计新的转录调控序列以指导外源基因在所选细胞中的治疗性表达。

The immunoglobulin mu heavy chain gene enhancer activates transcription and recombination in differentiating B lymphocytes. Enhancer activity is mediated by several DNA binding proteins which assemble into a precise multiprotein complex on the enhancer. The long-term objectives of this program is to understand the molecular mechanism of enhancer function. Two aspects of enhancer function will be addressed in this application. First, the mechanisms of combinatorial transcription control will be examined (Specific Aim 1). Combinatorial control refers to a central characteristic of enhancers, whereby the properties of an enhancer are very different from the properties of individual DNA binding proteins that interact with the enhancer. Biochemical and genetic experiments are proposed to understand the mechanisms by which distal sites on the enhancer cooperate to activate transcription and recombination. Second, the mechanism of chromatin structure modulation by the enhancer will be studied in-vitro (Specific Aim 2) and in B lymphocytes (Specific Aim 3). In-vitro chromatin assembly will be used to examine the effects of individual, and combinations, of mu enhancer binding proteins. Structural consequences on nucleosome positioning and chromatin structure will be assessed by DNAase I and micrococcal nuclease digestion assays. In parallel, functional consequences of chromatin re-structuring will be evaluated by in-vitro transcription and recombination assays. Lastly, the chromatin structure of the unrearranged IgH locus in pre-B cells will be mapped by DNAase I, micrococcal nuclease and histone acetylation. The limits of locus accessibility will be defined and the contribution of important regulatory elements will be determined by analyzing cells in which the sequences have been deleted. Taken together, these studies will provide comprehensive insights into the function of the mu enhancer. In-depth understanding of tissue-specific gene regulation will be essential to develop approaches to selectively reduce aberrant gene expression in diseased states and, conversely, to enhance gene expression to overcome immunodeficiencies. The rules of combinatorial transcription activation will also allow the design of novel transcription regulatory sequences to direct therapeutic expression of exogenous genes in selected cells.