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

项目摘要 背景现在很明显，大多数哺乳动物基因组具有表达非- 编码RNA（ncRNA）。然而，这些ncRNA的功能和调节机制仅仅是 开始被探索。生物学家遇到的一个挑战是检测这些ncRNA， 往往在转录上受到严格控制并迅速降解。使用鼠标模型， 为了检测lncRNA，我们最近鉴定了一组长的非编码RNA（lncRNA），它们表达于 围绕着免疫球蛋白基因座的基因。这些lncRNA被放置在拓扑关联的 在B细胞发育过程中形成的TADs。使用缺乏这些的小鼠模型进行的实验 lncRNA（已发表和初步数据）在类转换重组和体细胞重组中发挥作用 超突变机制 目标/假设。在这里，我们研究lncRNA在调节基因组局部表达中的直接作用。 结构和DNA拓扑结构通过顺式和反式机制。在目标1中：我们评估了 lncRNA表达位点通过组织TADIgH调节IgH重组;在目标2中，我们了解了 lncRNA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CSR中的功能。 研究设计：使用缺乏特异性lncRNA的小鼠模型和细胞系，我们旨在研究 免疫相关功能。为了评估lncRNA的分子作用机制，我们 使用生物化学分析来纯化lncRNA相互作用蛋白，并进行染色体结构分析 例如HiC和4C-seq。最后，我们进行了高通量测序实验，以评估 lncRNA在IG基因座基因和其他地方对SHM的影响。 疾病相关性：拟议的研究将有助于更好地了解B细胞中的机制。 发展和功能。本申请中研究的lncRNA在患者中携带多态性 伊加缺乏综合征，因此我们的研究与人类生理学有关。最后，抗体 多样化机制对于免疫系统的稳态是必不可少的，但当这些机制失败时， 与淋巴瘤相关的基因组改变增加。因此，本研究与 免疫力和淋巴瘤的发生

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.