Long Non-Coding RNAs in Allergy

过敏中的长非编码 RNA

基本信息

批准号：
10493379
负责人：
ADAM WILLIAMS
金额：
$ 57.46万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-23 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10493379
关键词：
Ablation Affinity Alleles Allergens Allergic Allergic Disease Alternaria Anaphylaxis Antibody Formation Antibody Response Antisense RNA Apoptotic Area Automobile Driving B-Lymphocytes BCL2L11 gene CD4 Positive T Lymphocytes CRISPR/Cas technology Cell Differentiation process Cell physiology Cells Chromatin Chromatin Interaction Analysis by Paired-End Tag Sequencing Chromatin Loop Communities Coupled DNA Data Dendritic Cells Elements Epidemic Epigenetic Process Gene Expression Gene Targeting Generations Genes Genetic Genetic Transcription Goals Health Helper-Inducer T-Lymphocyte Human Hypersensitivity IgE Immune response Immunity Immunization In Vitro Interleukin-13 Interleukin-4 Interleukin-5 Life Lung Maps Mass Spectrum Analysis Methodology Methods Modeling Molecular Mus PI3K/AKT Pathway interactions Phenotype Production Proteins RNA Regulation Regulator Genes Regulatory Element Regulatory Pathway Research Role Signal Transduction T cell response T-Lymphocyte Testing Th2 Cells Therapeutic Intervention Transcript Untranslated RNA Work allergic response base cell type cytokine eosinophil epigenetic silencing epigenome editing experimental study genetic regulatory protein genome wide association study genome-wide improved in vivo lung histology novel overexpression polarized cell promoter response single-cell RNA sequencing tRNA Precursor therapeutic target tool

项目摘要

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。