Defining the roles of an enhancer long non-coding RNA eIncRNA-ID2 in rickettsial pathogenesis and immunity

项目摘要 基因表达的精确和动态改变是调节宿主免疫的关键决定因素 微生物病原体。斑点热组中的致病性立克次体引起一些最严重的 人类感染性疾病，其特征是微血管炎症和功能障碍， 内皮细胞播散性感染和血管通透性增加，导致肺/脑 水肿长度大于等于200个核苷酸的长链非编码RNA通过以下途径调节一系列生物学反应： 一系列的机制和它们表达水平的变化现在错综复杂地联系在一起， 以及细胞介导的免疫反应。增强子lncRNA作为lncRNA的一个重要亚类， 通过以顺式或反式作用方式增强蛋白质编码基因（PCG）来实现其调节作用。 我们对肺进行了RNA测序，肺是易感性肺结核的主要受影响的靶器官之一。 小鼠感染R. conorii鉴定179个lncRNA的上调。通过随访分析， 基于启动子相关（plnc）RNA的单甲基化与三甲基化的比率， 赖氨酸4处的组蛋白3（H3 K4 Me 1：H3 K4 Me 3）和基于POLR 2A，p300， DNase I超敏位点、CTCF和Hi-3C ChIP-Seq数据集，我们进一步确定了显著高于 小鼠中活性elncRNA 013718及其靶点PCG DNA结合抑制剂2（ID 2）的表达 肺、脾和CD 8 + T细胞。我们的初步发现进一步表明， elncRNA 013718正调节E蛋白转录的蛋白质拮抗剂ID 2的表达 因子和免疫系统中T细胞的调节剂。因此，我们将elncRNA 013718称为elncRNA- ID 2和假设elncRNA-ID 2：Id 2在保护性宿主的调节中相互作用的新贡献作用 立克次体感染期间的免疫力。我们建议通过两个独立的主题来测试这一假设 相互关联的具体目标。目的1将区分elncRNA的细胞类型特异性表达和功能作用， ID 2和ID 2在宿主肺和脾中在实验鼠R. conorii和R.南方线虫感染 在目标2中，我们将确定对elncRNA的整体和细胞特异性干扰的调节作用。 ID 2对宿主免疫应答和疾病进展/结果的影响。鉴于细胞免疫的复杂性 反应，我们将采用两个独立的和建立的体内感染模型，密切模仿 人类立克次体病的病理生理学以及细胞微生物学和免疫学的前沿方法 为了确定elncRNA-ID 2作为一种新的elncRNA在以下分子回路中的调节潜力， 调节宿主免疫力。所获得的见解将增强我们对特定环境的生理学知识， elncRNA-ID 2在决定宿主对致病性立克次体应答中的作用，并可能揭示 独特的切入点，新的战略，以提高宿主免疫力对细胞内微生物感染。

PROJECT SUMMARY Precise and dynamic alterations in gene expression are critical determinants of the regulation of host immunity to microbial pathogens. Pathogenic rickettsiae in the spotted fever group cause some of the most severe infectious diseases in humans, characterized by microvascular inflammation and dysfunction attributed to disseminated infection of endothelial cells and increased vascular permeability resulting in pulmonary/cerebral edema. Long non-coding (lnc) RNAs of ≥ 200 nucleotides regulate a panoply of biological responses through an array of mechanisms and changes in their expression levels are now intricately linked to the determination of innate as well as cell-mediated immune responses. As an important subset of lncRNAs, enhancer lncRNAs implement their regulatory roles by enhancing protein coding genes (PCGs) in a cis- or trans-acting manner. We performed RNA-sequencing on the lungs as one of the predominantly affected target organs of susceptible mice infected with R. conorii to identify up-regulation of 179 lncRNAs. Via follow-up analysis to differentiate enhancer (elnc) from promoter-associated (plnc) RNAs based on the ratio of single- versus tri-methylation of histone 3 at lysine 4 (H3K4Me1:H3K4Me3) and other active enhancer signatures based on POLR2A, p300, DNase I hypersensitivity sites, CTCF, and Hi-3C ChIP-Seq datasets, we further determined significantly higher expression of an active elncRNA013718 and its target PCG Inhibitor of DNA binding 2 (ID2) in the mouse lungs, spleen, and CD8+ T-cells during Rickettsia conorii infection. Our preliminary findings further suggest that elncRNA013718 positively regulates the expression of ID2, a protein antagonist of E protein transcription factors and a regulator of T cells in the immune system. Accordingly, we refer to elncRNA013718 as elncRNA- ID2 and hypothesize novel contributory roles for elncRNA-ID2:Id2 interplay in the regulation of protective host immunity during rickettsial infections. We propose to test this hypothesis via two independent yet thematically interlinked specific aims. Aim 1 will distinguish cell type-specific expression and functional roles of elncRNA- ID2 and ID2 in the host lungs and spleen in experimental murine models of R. conorii and R. australis infection. In Aim 2, we will determine the modulatory effects of both global and cell-specific interference with elncRNA- ID2 on host immune responses and disease progression/outcome. Given the complexity of cellular immune responses, we will employ two independent and established in vivo models of infection closely mimicking the pathophysiology of human rickettsioses and cutting-edge approaches of cellular microbiology and immunology to determine the regulatory potential of elncRNA-ID2 as a novel elncRNA in the molecular circuitry underlying regulation of host immunity. The acquired insights will enhance our knowledge of context-specific physiological roles of elncRNA-ID2 in the determination of host responses to pathogenic rickettsiae and reveal potentially unique entry points for novel strategies to enhance host immunity against intracellular microbial infections.