Long noncoding RNA expressing genomic elements that control antibody diversification and chromosomal integrity in B cells

项目总结 背景资料。现在很明显，哺乳动物的大多数基因组都有可能表达非 编码RNA(NcRNAs)。然而，这些ncRNA的调控功能和机制(S)只是 开始被探索。生物学家遇到的一个挑战是检测这些ncRNA，这是 往往在转录上受到严格控制，并迅速降解。使用鼠标模型，可以轻松实现 检测lncRNA，我们最近发现了一组表达的长非编码RNA(LncRNA) 围绕着免疫球蛋白基因。这些lncRNA被放置在拓扑关联的内部 B细胞发育过程中形成的结构域(TADs)。使用缺乏这些功能的小鼠模型进行的实验 LncRNAs(已发表的和初步的数据)展示了在类交换重组和体细胞中的作用 超突变机制。 目标/假设。在这里，我们研究了lncRNAs在基因组局部调控中的直接作用。 通过顺式和反式机制的体系结构和DNA拓扑。在目标1中：我们评估了 通过组织TADIgH调节IgH重组中的lncRNA表达位点；在目标2中，我们了解 LncRNAs被用于控制基因组结构的分子机制；在目标3中，我们 重点了解lncRNA SµGLT在控制企业社会责任中的作用。 研究设计：利用缺乏特定lncRNAs的小鼠模型和细胞系，我们旨在研究 它们的免疫相关功能。为了评估lncRNAs的分子作用机制，我们 用生化方法提纯lncRNA相互作用蛋白并进行染色体构型分析 例如HIC和4C-Seq。最后，我们进行了高通量测序实验，以评估 LncRNA对免疫球蛋白基因和其他部位SHM的影响(S)。 疾病相关性：拟议的研究将有助于更好地理解B细胞的机制 发展和功能。在这一应用中研究的lncRNAs在患者中携带多态 患有IgA缺乏综合症，因此我们的研究与人类生理学有关。最后，抗体 多样化机制对免疫系统动态平衡是必不可少的，但当这些机制失效时 与淋巴瘤相关的基因组改变增加。因此，这项研究与 免疫和淋巴肿大都有。

PROJECT SUMMARY Background. It is now evident that the majority of the mammalian genome has the potential to express non- coding RNAs (ncRNAs). However, the functionality and mechanism(s) of regulation of these ncRNAs are just beginning to be explored. One challenge that biologists encounter is the detection of these ncRNAs, which often tend to be transcriptionally tightly controlled and rapidly degraded. Using mouse models that allow easy detection of lncRNA, we have recently identified a set of long noncoding RNAs (lncRNA) that are expressed surrounding the immunoglobulin loci genes. These lncRNAs are placed inside topologically associating domains (TADs) that are formed during B cell development. Experiments using mouse models that lack these lncRNAs (published and preliminary data) demonstrate roles in class switch recombination and somatic hypermutation mechanisms. Objectives/Hypothesis. Here we investigate the direct role of lncRNAs in regulating genome local architecture and DNA topology via cis and trans mechanisms. In aim 1: we evaluate the physiological role of lncRNA-expressing loci in regulating IgH recombination via organizing TADIgH; in aim 2, we understand the molecular mechanisms through which lncRNAs are used to control genome architecture; and in aim 3, we focus on understanding the function of lncRNA SµGLT in control of CSR. Study Design: Using mouse models and cell lines that are deficient in specific lncRNAs we aim to investigate their immunologically relevant functions. For evaluating the molecular mechanism of action of the lncRNAs we use biochemical assays to purify lncRNA interacting proteins and perform chromosomal architecture assays such as HiC and 4C-seq. Finally, we perform high-throughput sequencing experiments to evaluate the lncRNA's effect(s) on SHM in the Ig loci genes and elsewhere. Disease Relevance: The proposed studies will lead to a better understanding of the mechanisms in B cell development and function. The lncRNAs being investigated in this application carry polymorphisms in patients with IgA deficiency syndrome and thus our study is relevant for human physiology. Finally, antibody diversification mechanisms are essential for immune system homeostasis but when these mechanisms fail there are increased genomic alterations that are associated with lymphomas. Thus, this study is related with both immunity and lymphomagenesis.