Non-coding RNAs in B lymphocyte development and function

类转换对免疫球蛋白基因重组和进一步多样化的调控 重组和体细胞超突变在细胞类型、特定阶段和等位基因中受到严格控制 具体的方式。调节抗原受体基因染色质可及性的精确机制 在这些事件中，人们仍然难以捉摸。我们的目标是评估RNAi在B细胞发育和 功能。我们之前已经证明了miRNAs对于B细胞的生存是必不可少的。具体来说， Dird、DROSHA或Dgcr8缺失后B细胞中miRNAs的消融阻断了Pro-B到Pre-B细胞的转变 由于细胞凋亡增加而导致的过渡。通过使用支持生存的bcl2挽救B细胞的发育 转基因，我们能够研究短的非编码RNA在V(D)J重组中的作用以及 在B细胞分化的后期。这些研究的初步数据显示，消融迪切尔 影响初级免疫系统，当DROSHA在类似的 方式，这表明依赖于DICER的非编码RNA在免疫球蛋白的直接调节中发挥作用 精神错乱。我们将继续研究这些非编码RNA的性质和作用，并研究 RNAi在生发中心反应中对免疫球蛋白基因座可及性的贡献。这个 依赖DICER的微处理器复合体非依赖的RNAi对免疫球蛋白基因座的控制 机制代表了哺乳动物基因座调控的一种新机制。我们的目标是了解 在此背景下，对染色质可及性的调节。此外，挽救B细胞发育的能力 在没有RNAi的情况下，我们有机会研究非编码RNA在B细胞终末的作用 分化和miRNA在浆细胞生成中的作用--作为对终末B细胞的调节 差异化计划仍然知之甚少。 我们的工作因超级风暴桑迪而遭受重大打击，因为所有用于这些的动物模型 随着动物设施的洪水泛滥，研究工作也随之丢失。此外，我们的实验室被迫搬迁 暴风雨过后，我们的建筑遭到严重破坏。此应用程序概述了我们在以下方面所做的努力 恢复我们研究所需的复杂动物模型，并复制令人兴奋的 我们需要在未来一到两年内提交具有竞争力的RO1申请的初步数据 好几年了。具体地说，我们将从储存的骨骼中分离的B细胞中产生可诱导的多能细胞(IPS 我们的原始小鼠的骨髓样本为这项研究产生了必要的小鼠模型。这个 使用B细胞来源的iPS细胞的方法将概括我们的 能够研究非编码RNA在B细胞发育后期的作用，并为我们提供 研究非编码RNA在体细胞超突变和类开关重组中的作用的机会 在几个独特重排的免疫球蛋白基因座的背景下。

Regulation of recombination and further diversification of immunoglobulin genes by class switch recombination and somatic hypermutation is tightly controlled in a cell type, stage specific and allele specific manner. The precise mechanisms that regulate chromatin accessibility of antigen receptor genes during these events remain elusive. Our goal is to evaluate the role of RNAi in B cell development and function. We have previously demonstrated that miRNAs are essential for B cell survival. Specifically, ablation of miRNAs in B cells upon deletion of Dicer, Drosha or DGCR8 blocked the pro-B to pre-B cell transition due to an increase in apoptosis. By rescuing B cell development using a pro-survival Bcl2 transgene, we are able to investigate the role of short non-coding RNAs in V(D)J recombination as well as in later stages of B cell differentiation. Preliminary data from these studies reveals that ablation of Dicer impacts primary immune repertoire and that this does not happen when Drosha is deleted in a similar manner, suggesting a role for Dicer dependent non-coding RNAs in direct regulation of immunoglobulin loci. We will extend our studies to investigate the nature and role of these non-coding RNAs and examine the contribution of RNAi to immunoglobulin locus accessibility during the germinal center reaction. The control of the immunoglobulin loci by Dicer dependent, microprocessor complex independent RNAi mechanism represents a novel mechanism in mammalian locus regulation. Our goal is to understand the regulation of chromatin accessibility in this context. In addition, the ability to rescue B cell development in the absence of RNAi gives us an opportunity to examine the role of non-coding RNAs in terminal B cell differentiation and the role of miRNA in plasma cell generation – as the regulation of the terminal B cell differentiation program remains poorly understood. Our work suffered a significant hit as a result of superstorm Sandy as all the animal models used for these studies were lost with the flooding of the animal facility. In addition, our laboratory was forced to relocate following the storm due to significant damage to our building. This application outlines our efforts to recover the complex animal models necessary for our research and to reproduce the exciting preliminary data that we need to submit a competitive RO1 application within the next one to two years. Specifically, we will generate inducible pluripotent cells (iPS) from the sorted B cells of stored bone marrow samples of our original mice to generate the necessary mouse models for this research. The approach of using B cell derived iPS cells with conditional Dicer and DGCR8 alleles will recapitulate our ability to investigate the role of non-coding RNAs in the later stages of B cell development and give us an opportunity to study the role of non-coding RNAs in somatic hypermutation and class switch recombination in the context of several uniquely rearranged immunoglobulin loci.