Non-coding RNAs in B lymphocyte development and function

通过类别转换调节免疫球蛋白基因的重组和进一步多样化 重组和体细胞超突变在细胞类型、阶段特异性和等位基因中受到严格控制。 具体方式。调节抗原受体基因染色质可及性的精确机制 在这些事件中仍然难以捉摸。我们的目标是评估RNAi在B细胞发育中的作用， 功能我们先前已经证明了miRNA对于B细胞存活是必不可少的。具体地说， Dicer、Drosha或DGCR 8缺失后，B细胞中miRNA的消融阻断了前B细胞向前B细胞的转化 由于凋亡的增加而转变。通过使用促存活Bcl 2挽救B细胞发育， 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， 在B细胞分化的后期阶段。这些研究的初步数据显示，切除Dicer 影响初级免疫库，而当Drosha在类似的免疫系统中缺失时， Dicer依赖性非编码RNA在免疫球蛋白的直接调节中的作用 的位点我们将扩大我们的研究，以调查这些非编码RNA的性质和作用，并检查 RNAi对免疫球蛋白基因座可及性的贡献。的 通过Dicer依赖性、微处理器复合物独立性RNAi控制免疫球蛋白基因座 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 在这种情况下对染色质可及性的调节。此外，拯救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在末端B细胞中的作用。 分化和miRNA在浆细胞生成中的作用-作为终末B细胞的调节 差异化方案仍然知之甚少。 由于超级风暴桑迪，我们的工作遭受了重大打击，因为所有用于这些的动物模型 动物设施被水淹没，研究也随之消失。此外，我们的实验室被迫搬迁 由于我们的建筑物受到严重破坏，本申请概述了我们的努力， 恢复我们研究所需的复杂动物模型，并重现令人兴奋的 我们需要在接下来的一到两个月内提交具有竞争力的RO 1申请的初步数据 年具体来说，我们将从储存的骨中分选出B细胞，产生诱导性多能细胞（iPS 我们的原始小鼠的骨髓样本，以产生本研究所需的小鼠模型。的 使用具有条件Dicer和DGCR 8等位基因的B细胞衍生的iPS细胞的方法将概括我们的研究。 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 有机会研究非编码RNA在体细胞超突变和类别转换重组中的作用 在几个独特重排的免疫球蛋白基因座的情况下。

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.