Long Non-Coding RNAs in Allergy

项目摘要 这项建议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）2型过敏反应的特征是产生CD 4 + T细胞， 辅助性2型（Th 2）细胞，以及最近鉴定的IL-13+滤泡辅助性T细胞（Tfh 13），其驱动生产 高亲和力过敏性IgE鉴于它们在过敏中的核心作用，了解T细胞如何编程 成为Th 2和Tfh 13细胞可以允许操纵T细胞应答以减轻过敏。最近的工作已经 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， 治疗干预的新目标和途径。lncRNA不编码蛋白质;相反，许多lncRNA产生 功能性RNA转录物是细胞身份、功能和存活的强大调节剂。我们的初步 数据显示lncRNA Morrbid控制CD 4 + T细胞功能，并且是2型免疫应答所需的 in vivo. CD 4 + T细胞的单细胞RNA测序（scRNA-Seq）显示Morrbid在CD 4 + T细胞中表达最高。 在表达IL 13的Th 2和Tfh 13细胞中，在Morrbid-/-小鼠的2型应答期间Tfh 13细胞减少。 我们的假设是，Morrbid是过敏反应过程中Th 2和Tfh 13分化的表观遗传调节因子。 应答在目标1中，我们将确定需要Morrbid产生2型免疫应答的细胞类型。使用 将条件性Morrbid等位基因与不同Cre表达系杂交的小鼠，我们将测试Morrbid的功能 在体内2型应答期间树突状细胞、B细胞、T细胞和Tfh细胞中。我们将分析 使用scRNA-Seq在来自具有或不具有过敏症的供体的T细胞中检测人MORBID，以确定Th 2 而Tfh细胞在变态反应中过表达MORBID。最后，使用基于CRISPR/Cas9的表观基因组编辑， 在人T细胞中，我们将确定MORBID沉默和过表达对CD 4 + T辅助细胞的影响， 体外极化。在目标2中，我们将剖析Morrbid位点调控基因的分子机制 在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 处理以消除lncRNA转录物而不阻断体内转录。在目标3中，我们将绘制Morrbid 相互作用组以确定Morrbid如何控制T细胞功能。为了识别与Morrbid直接结合的基因，我们 将采用一种新的方法，允许同时映射lncRNA-染色质相互作用， lncRNA相关的染色质环全基因组，称为RNA ChIA-PET（RNA-染色质相互作用分析 通过配对末端标签测序）。为了鉴定与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）。通过实现这些目标，我们将 阐明了控制2型免疫的新的调节途径，这些途径可能被用于治疗过敏。 此外，我们将建立并验证两个对研究界有重大益处的新工具 其缺乏分析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。

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.