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

项目概要 背景。现在很明显，大多数哺乳动物基因组都有可能表达非 编码 RNA (ncRNA)。然而，这些 ncRNA 的功能和调控机制尚不明确。 开始被探索。生物学家遇到的一项挑战是这些 ncRNA 的检测， 通常往往受到转录严格控制并迅速降解。使用易于操作的鼠标模型 lncRNA的检测，我们最近鉴定了一组表达的长非编码RNA（lncRNA） 围绕免疫球蛋白位点基因。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 (TAD)。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）展示了类别转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lncRNA在调节基因组局部中的直接作用 通过顺式和反式机制的结构和 DNA 拓扑。目标 1：我们评估以下物质的生理作用： lncRNA表达位点通过组织TADIgH调节IgH重组；在目标 2 中，我们了解 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：我们旨在使用缺乏特定 lncRNA 的小鼠模型和细胞系进行研究 它们的免疫学相关功能。为了评估 lncRNA 的分子作用机制，我们 使用生化测定来纯化 lncRNA 相互作用蛋白并进行染色体结构测定 例如 HiC 和 4C-seq。最后，我们进行高通量测序实验来评估 lncRNA 对 Ig 位点基因和其他地方的 SHM 的影响。 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 发育和功能。本申请中正在研究的 lncRNA 在患者中携带多态性 患有 IgA 缺乏综合症，因此我们的研究与人类生理学相关。最后是抗体 多样化机制对于免疫系统稳态至关重要，但当这些机制失败时 与淋巴瘤相关的基因组改变有所增加。因此，本研究与 免疫和淋巴瘤发生。

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.