Characterization of Chromatin associated Long non-coding in immunity

描述（由申请人提供）：先天免疫应答通过驱动炎症、宿主防御途径和适应性免疫来对抗感染性微生物。不同功能类别内的多个基因通过转录“开”和“关”开关协调地和暂时地调节，所述转录“开”和“关”开关解释响应于微生物触发物的基因表达的特异性。明确定义的DNA结合转录因子、转录辅助调节因子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也有助于在不同的生物背景下的基因调控。越来越多的证据，包括我们自己发表的研究，支持它们在免疫系统中的重要性。在巨噬细胞、树突细胞、嗜中性粒细胞、T细胞和B细胞的分化和/或活化过程中，已在它们中鉴定出lncRNA。我们已经发现这些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相关，以调节免疫基因的转录。我们建议在静息和活化的人类巨噬细胞中识别和分类染色质相关lncRNA，并采用尖端分子方法来功能性地表征这些调节剂的基因组靶点。 这些目标将通过两个具体目标来实现：在目标1中，我们将使用蔗糖密度梯度分级以及染色质免疫沉淀结合RNA测序以无偏的方式在人巨噬细胞中鉴定染色质相关lncRNA（ca-lncRNA）。然后将使用Nanostring技术分析这些ca-lncRNA的调节，以评估响应微生物产物、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aim 2中，我们将分别使用RNA反义纯化（RAP）结合DNA测序或质谱法鉴定染色质相关lncRNA的基因组靶点和蛋白质结合伴侣。这些研究将定义在染色质水平上起作用以调节巨噬细胞转录反应的功能重要的lncRNA，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。这种功能相关lncRNA的无偏鉴定也将为未来更详细地表征这些基因的研究提供平台。它们还将为在其他生物学背景下研究lncRNA提供新的策略。

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.