Long Non-Coding RNAs in Allergy
过敏中的长非编码 RNA
基本信息
- 批准号:10363445
- 负责人:
- 金额:$ 18.94万
- 依托单位:
- 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 2022-01-31
- 项目状态:已结题
- 来源:
- 关键词:AblationAffinityAllelesAllergensAllergicAllergic DiseaseAlternariaAnaphylaxisAntibody FormationAntibody ResponseAntisense RNAApoptoticAreaAutomobile DrivingB-LymphocytesBCL2L11 geneCD4 Positive T LymphocytesCRISPR/Cas technologyCell Differentiation processCell physiologyCellsChromatinChromatin Interaction Analysis by Paired-End Tag SequencingChromatin LoopCommunitiesCoupledDNADataDendritic CellsElementsEpidemicEpigenetic ProcessGene ExpressionGene TargetingGenerationsGenesGeneticGenetic TranscriptionGoalsHealthHelper-Inducer T-LymphocyteHistologyHumanHypersensitivityIgEImmune responseImmunityImmunizationIn VitroInterleukin-13Interleukin-4Interleukin-5LifeLungMapsMass Spectrum AnalysisMethodologyMethodsModelingMolecularMusPI3K/AKTPathway interactionsPhenotypeProductionProteinsRNARegulationRegulator GenesRegulatory ElementRegulatory PathwayResearchRoleSignal TransductionT cell responseT-LymphocyteTestingTh2 CellsTherapeutic InterventionTranscriptUntranslated RNAWorkallergic responsebasecell typecytokineeosinophilepigenetic silencingepigenome editingexperimental studygenetic regulatory proteingenome wide association studygenome-wideimprovedin vivonoveloverexpressionpolarized cellpromoterresponsesingle-cell RNA sequencingtRNA Precursortherapeutic targettool
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ADAM WILLIAMS其他文献
ADAM WILLIAMS的其他文献
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{{ truncateString('ADAM WILLIAMS', 18)}}的其他基金
lncRNA Control of Airway Epithelial Cell Responses to Type 2 Inflammation
lncRNA 控制气道上皮细胞对 2 型炎症的反应
- 批准号:
10318082 - 财政年份:2019
- 资助金额:
$ 18.94万 - 项目类别:
lncRNA Control of Airway Epithelial Cell Responses to Type 2 Inflammation
lncRNA 控制气道上皮细胞对 2 型炎症的反应
- 批准号:
10555004 - 财政年份:2019
- 资助金额:
$ 18.94万 - 项目类别:
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