Characterization of Chromatin associated Long non-coding in immunity

免疫中染色质相关长非编码的表征

• 批准号: 8809301
• 负责人: Katherine A. Fitzgerald
• 金额: $ 23.94万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8809301
• 关键词: Accounting; Address; Air; Antisense RNA; Arthritis; Atherosclerosis; Automobile Driving; B-Lymphocytes; Binding; Binding Sites; Biological; Biological Process; Cataloging; Catalogs; Categories; Cells; Chromatin; Code; Collection; Communities; Complex; Coupled; DNA Binding; DNA Sequence; Dendritic Cells; Density Gradient Centrifugation; Disease; Event; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Guilt; Heart Diseases; High-Throughput DNA Sequencing; Host Defense; Human; Immune; Immune response; Immune system; Immunity; Inflammation; Inflammatory; Invaded; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Microbe; Molecular; Neoplasm Metastasis; Nuclear Import; Nucleotides; Pathway interactions; Positioning Attribute; Protein Binding; Proteins; Publishing; RNA; RNA Sequences; Regulation; Research Design; Rest; Signal Transduction; Site; Specificity; Sucrose; T-Lymphocyte; Technology; Tissues; Untranslated RNA; Vaccines; Viral; Work; X Inactivation; adaptive immunity; base; chromatin immunoprecipitation; combat; genome-wide; imprint; macrophage; meetings; microbial; nano-string; neutrophil; pathogen; prevent; public health relevance; response; screening; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): Innate immune responses combat infectious microbes by driving inflammation, host-defense pathways and adaptive immunity. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional "on" and "off" switches that account for the specificity of gene expression in response to microbial triggers. Well-defined DNA binding transcription factors, transcriptional co-regulators and chromatin modifying complexes collaborate to coordinate transcription of immune genes. Recent evidence indicates that non-coding RNAs also contribute to gene regulation in diverse biological contexts. A growing body of evidence including our own published studies supports their importance in the immune system. lncRNAs have been identified in macrophages, dendritic cells, neutrophils, T-cells and B-cells during their differentiation and/or activation. We have found that these lncRNAs in turn regulate expression of other immune genes. This proposal is based on our hypothesis that lncRNAs are associated with chromatin in immune cells in order to regulate transcription of immune genes. We propose to identify and catalogue chromatin-associated lncRNAs in both resting and activated human macrophages and employ cutting edge molecular approaches to functionally characterize the genomic targets of these regulators. These objectives will be met using two specific aims: In aim 1 we will identify chromatin-associated lncRNAs (ca-lncRNAs) in an unbiased manner in human macrophages using sucrose density gradient fractionation as well as chromatin Immunoprecipitation coupled to RNA-sequencing. The regulation of these ca-lncRNAs will then be profiled using Nanostring technology to assess the temporal regulation and signaling mechanisms regulating their expression in response to microbial products, viral and bacterial pathogens. In aim2 we will identify the genomic targets and protein-binding partners of chromatin-associated lncRNAs using RNA antisense purification (RAP) coupled to DNA-Sequencing or Mass Spectrometry, respectively. These studies will define functionally important lncRNAs acting at the level of chromatin to modulate macrophage transcriptional responses and define genomic targets of ca-lncRNAs regulated during host-pathogen interactions. This unbiased identification of functionally relevant lncRNAs will also provide a platform for future studies that characterize these genes in greater detail. They will also provide new strategies to study lncRNA in other biological contexts.

描述（由申请人提供）：先天免疫反应通过驱动炎症，宿主防御途径和适应性免疫来对抗传染性微生物。不同功能类别中的多个基因由转录“ ON”和“ OFF”开关进行协调和时间调节，这些开关响应了对微生物触发器的基因表达的特异性。定义明确的DNA结合转录因子，转录共调控剂和染色质修饰复合物协作以协调免疫基因的转录。最近的证据表明，非编码RNA在不同的生物学环境下也有助于基因调节。包括我们自己已发表的研究在内的越来越多的证据支持它们在免疫系统中的重要性。 LNCRNA在分化和/或激活过程中已经在巨噬细胞，树突状细胞，中性粒细胞，T细胞和B细胞中鉴定出来。我们发现这些LNCRNA反过来调节其他免疫基因的表达。该建议基于我们的假设，即LNCRNA与免疫细胞中的染色质有关，以调节免疫基因的转录。我们建议在静止和激活的人类巨噬细胞中识别和分类与染色质相关的LNCRNA，并采用尖端分子方法来在功能上表征这些调节剂的基因组靶标。 这些目标将使用两个特定的目标实现：在AIM 1中，我们将在人类巨噬细胞中使用蔗糖密度梯度分馏以及染色质免疫沉淀在人类巨噬细胞中以公正的方式鉴定与染色质相关的LNCRNA（CA-LNCRNA）。然后，将使用纳米串技术对这些CA-LNCRNA的调节进行介绍，以评估对微生物产物，病毒和细菌病原体调节其表达的时间调节和信号传导机制。在AIM2中，我们将分别使用与DNA测序或质谱法的RNA反义纯化（RAP）（RAP）确定与染色质相关的LNCRNA的基因组靶标和蛋白质结合伴侣。这些研究将定义作用在染色质水平的功能上重要的LNCRNA，以调节巨噬细胞转录反应，并定义在宿主 - 病原体相互作用期间调节的CA-LNCRNA的基因组靶标。对功能相关的LNCRNA的这种公正的识别还将为将来的研究提供一个平台，以更详细地表征这些基因。他们还将提供在其他生物学环境中研究lncRNA的新策略。