Long Non-Coding RNAs in Allergy

项目总结 这项建议的总体目标是确定控制过敏反应的基本机制。 非编码RNA(LncRNA)Morrbid。2型过敏反应的特征是产生CD4T 辅助型2(Th2)细胞，以及最近发现的IL-13 T滤泡辅助性(Tfh13)细胞，它们驱动产生 高亲和力过敏性免疫球蛋白。鉴于T细胞在过敏中的核心作用，了解T细胞是如何编程的 成为Th2和Tfh13细胞可以通过操纵T细胞的反应来减轻过敏。最近的工作有 揭示了lncRNAs在免疫中的关键作用，开辟了一个令人兴奋的研究领域，可能会发现 治疗干预的新靶点和新途径。LncRNA不编码蛋白质；相反，许多lncRNA产生 功能RNA转录本，是细胞身份、功能和生存的强大调节者。我们的预赛 数据显示，lncRNA Morrid控制着CD4T细胞的功能，是2型免疫反应所必需的 在活体内。CD4T细胞的单细胞RNA测序(scRNA-Seq)显示Morrid的表达最高 在表达IL13的Th2和Tfh13细胞中，在Morrid-/-小鼠的2型反应中，Tfh13细胞减少。 我们的假设是，Morrid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，我们将确定需要MorrBid来产生2型免疫反应的细胞类型。vbl.使用 带有条件Morrid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 树突状细胞、B细胞、T细胞和Tfh细胞在体内的2型反应。我们将分析 用scRNA-Seq检测有无过敏反应的供者T细胞中的人MORRBID，以确定Th2 TFH细胞在变态反应中过度表达MORRBID。最后，使用基于CRISPR/Cas9的初步表观基因组编辑 人类T细胞，我们将确定MORRBID沉默和过度表达对CD4T辅助细胞的影响 体外极化。在目标2中，我们将剖析Morrbi基因座调控基因的分子机制。 在T细胞中表达。为此，我们开发了一种新的基于pre-tRNA的基因靶向策略 在体内去除IncRNA转录本而不阻止转录的处理。在目标3中，我们将绘制莫尔比特 相互作用组以确定Morrid如何控制T细胞功能。为了识别与莫尔比特直接结合的基因，我们 将使用一种新的方法，允许同时映射LncRNA-染色质相互作用和 与LncRNA相关的染色质在全基因组范围内循环，称为RNA Chia-PET(RNA-染色质相互作用分析 通过成对末端标签测序)。为了鉴定与Morrid相互作用的调控蛋白，我们将使用RAP-MS (RNA反义纯化与质谱仪联用)。通过实现这些目标，我们将 阐明控制2型免疫的新的调控途径，这些途径可能被用于治疗过敏。 此外，我们将建立和验证两个对研究界有重大好处的新工具 缺乏分析lncRNA功能的标准方法；lncRNA特异的基因- 靶向方法，以及一种绘制全基因组lncRNA相关染色体相互作用图的方法。

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.