Long Non-Coding RNAs in Allergy

过敏中的长非编码 RNA

• 批准号: 10555000
• 负责人: ADAM WILLIAMS
• 金额: $ 63.15万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-23 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10555000
• 关键词: Long; Non; Coding; RNAs; Allergy

PROJECT SUMMARY The overall goal of this proposal is to identify fundamental mechanisms controlling allergic responses by the long non-coding RNA (lncRNA) Morrbid. Type 2 allergic responses are characterized by the generation of CD4+ T helper type 2 (Th2) cells, and the recently identified IL-13+ T follicular helper (Tfh13) cells, which drive production of high-affinity anaphylactic IgE. Given their central role in allergy, understanding how T cells are programmed to become Th2 and Tfh13 cells could allow manipulation of T cell responses to mitigate allergy. Recent work has revealed a critical function for lncRNAs in immunity, opening up an exciting area of research that may uncover new targets and pathways for therapeutic intervention. lncRNAs do not encode proteins; rather, many produce functional RNA transcripts that are powerful regulators of cellular identity, function and survival. Our preliminary data show that the lncRNA Morrbid controls CD4+ T cell function and is required for type 2 immune responses in vivo. Single-cell RNA-sequencing (scRNA-Seq) of CD4+ T cells revealed Morrbid to be most highly expressed in Il13-expressing Th2 and Tfh13 cells, and Tfh13 cells are reduced during type 2 responses in Morrbid-/- mice. Our hypothesis is that Morrbid is an epigenetic regulator of Th2 and Tfh13 differentiation during allergic responses. In Aim 1 we will identify cell types that require Morrbid to generate type 2 immune responses. Using mice with a conditional Morrbid allele crossed to different Cre-expressing lines, we will test the function of Morrbid in dendritic cells, B cells, T cells, and Tfh cells during type 2 responses in vivo. We will analyze expression of human MORRBID in T cells from donors with or without allergies using scRNA-Seq, to determine whether Th2 and Tfh cells overexpress MORRBID in allergy. Finally, using CRISPR/Cas9-based epigenome editing in primary human T cells, we will determine the impact of MORRBID silencing and overexpression on CD4+ T helper cell polarization in vitro. In Aim 2 we will dissect molecular mechanisms by which the Morrbid locus regulates gene expression in T cells. To this end we have developed a novel genetic targeting strategy based on pre-tRNA processing to ablate lncRNA transcripts without blocking transcription in vivo. In Aim 3 we will map the Morrbid interactome to determine how Morrbid controls T cell function. To identify genes directly bound by Morrbid, we will employ a novel method that allows simultaneous mapping of both lncRNA-chromatin interactions and lncRNA-associated chromatin loops genome-wide, called RNA ChIA-PET (RNA-Chromatin Interaction Analysis by Paired-End Tag sequencing). To identify regulatory proteins interacting with Morrbid, we will use RAP-MS (RNA Antisense Purification coupled with Mass Spectrometry). Through completion of these Aims, we will elucidate new regulatory pathways controlling type 2 immunity that could potentially be exploited to treat allergy. In addition, we will have established and validated two novel tools of significant benefit to the research community which suffers from a dearth of standard methodology for analyzing lncRNA function; a lncRNA-specific gene- targeting approach, and a method to map genome-wide lncRNA-associated chromosomal interactions.

项目概要 该提案的总体目标是确定长期控制过敏反应的基本机制 非编码 RNA (lncRNA) 病态。 2 型过敏反应的特点是产生 CD4+ T 2 型辅助细胞 (Th2) 和最近发现的 IL-13+ T 滤泡辅助细胞 (Tfh13) 细胞可驱动生产 高亲和力过敏性 IgE。鉴于 T 细胞在过敏中的核心作用，了解 T 细胞的编程方式 变成 Th2 和 Tfh13 细胞可以控制 T 细胞反应以减轻过敏。最近的工作有 揭示了 lncRNA 在免疫中的关键功能，开辟了一个令人兴奋的研究领域，可能会揭示 治疗干预的新目标和途径。 lncRNA不编码蛋白质；相反，许多生产 功能性 RNA 转录本是细胞身份、功能和生存的强大调节剂。我们的初步 数据显示，lncRNA Morrbid 控制 CD4+ T 细胞功能，并且是 2 型免疫反应所必需的 体内。 CD4+ T 细胞的单细胞 RNA 测序 (scRNA-Seq) 显示 Morrbid 表达最高 在表达 Il13 的 Th2 和 Tfh13 细胞中，Tfh13 细胞在 Morrbid-/- 小鼠的 2 型反应期间减少。 我们的假设是 Morrbid 是过敏过程中 Th2 和 Tfh13 分化的表观遗传调节因子 回应。在目标 1 中，我们将鉴定需要 Morrbid 才能产生 2 型免疫反应的细胞类型。使用 将具有条件 Morrbid 等位基因的小鼠与不同的 Cre 表达系杂交，我们将测试 Morrbid 的功能 在体内 2 型反应期间的树突状细胞、B 细胞、T 细胞和 Tfh 细胞中。我们将分析表达式 使用 scRNA-Seq 对来自有或没有过敏的捐赠者的 T 细胞中的人 MORRBID 进行检测，以确定 Th2 是否存在 Tfh 细胞在过敏中过度表达 MORRBID。最后，在初级中使用基于 CRISPR/Cas9 的表观基因组编辑 人类 T 细胞，我们将确定 MORRBID 沉默和过度表达对 CD4+ T 辅助细胞的影响 体外极化。在目标 2 中，我们将剖析 Morrbid 基因座调节基因的分子机制 T 细胞中的表达。为此，我们开发了一种基于前tRNA的新型遗传靶向策略 加工消除 lncRNA 转录本而不阻断体内转录。在目标 3 中，我们将绘制病态地图 相互作用组以确定 Morrbid 如何控制 T 细胞功能。为了鉴定与 Morrbid 直接结合的基因，我们 将采用一种新方法，可以同时绘制 lncRNA-染色质相互作用和 全基因组范围内的 lncRNA 相关染色质环，称为 RNA ChIA-PET（RNA-染色质相互作用分析） 通过双端标签测序）。为了识别与 Morrbid 相互作用的调节蛋白，我们将使用 RAP-MS （RNA 反义纯化与质谱联用）。通过完成这些目标，我们将 阐明控制 2 型免疫的新调控途径，可用于治疗过敏。 此外，我们将建立并验证两种对研究界有重大益处的新颖工具 缺乏分析 lncRNA 功能的标准方法； lncRNA特异性基因- 靶向方法，以及绘制全基因组 lncRNA 相关染色体相互作用的方法。