Table_3_Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection.DOC

Recent discovery that much of the mammalian genome does not encode protein-coding genes (PCGs) has brought widespread attention to long noncoding RNAs (lncRNAs) as a novel layer of biological regulation. Enhancer lnc (elnc) RNAs from the enhancer regions of the genome carry the capacity to regulate PCGs in cis or in trans. Spotted fever rickettsioses represent the consequence of host infection with Gram-negative, obligate intracellular bacteria in the Genus Rickettsia. Despite being implicated in the pathways of infection and inflammation, the roles of lncRNAs in host response to Rickettsia species have remained a mystery. We have profiled the expression of host lncRNAs during infection of susceptible mice with R. conorii as a model closely mimicking the pathogenesis of human spotted fever rickettsioses. RNA sequencing on the lungs of infected hosts yielded reads mapping to 74,964 non-coding RNAs, 206 and 277 of which were determined to be significantly up- and down-regulated, respectively, in comparison to uninfected controls. Following removal of short non-coding RNAs and ambiguous transcripts, remaining transcripts underwent in-depth analysis of mouse lung epigenetic signatures H3K4Me1 and H3K4Me3, active transcript markers (POLR2A, p300, CTCF), and DNaseI hypersensitivity sites to identify two potentially active and highly up-regulated elncRNAs NONMMUT013718 and NONMMUT024103. Using Hi-3C sequencing resource, we further determined that genomic loci of NONMMUT013718 and NONMMUT024103 might interact with and regulate the expression of nearby PCGs, namely Id2 (inhibitor of DNA binding 2) and Apol10b (apolipoprotein 10b), respectively. Heterologous reporter assays confirmed the activity of elncRNAs as the inducers of their predicted PCGs. In the lungs of infected mice, expression of both elncRNAs and their targets was significantly higher than mock-infected controls. Induced expression of NONMMUT013718/Id2 in murine macrophages and NONMMUT024103/Apol10b in endothelial cells was also clearly evident during R. conorii infection in vitro. Finally, shRNA mediated knock-down of NONMMUT013718 and NONMMUT024103 elncRNAs resulted in reduced expression of endogenous Id2 and Apl10b, demonstrating the regulatory roles of these elncRNAs on their target PCGs. Our results provide very first experimental evidence suggesting altered expression of pulmonary lncRNAs and elncRNA-mediated regulation of PCGs involved in immunity and during host interactions with pathogenic rickettsiae.

近期研究发现，哺乳动物基因组中绝大多数区域并不编码蛋白编码基因（protein-coding genes, PCGs），这使得长非编码RNA（long noncoding RNAs, lncRNAs）作为一类全新的生物调控层受到广泛关注。源自基因组增强子区域的增强子长非编码RNA（enhancer lnc, elnc RNAs）具备顺式或反式调控蛋白编码基因的能力。
斑点热立克次体病（Spotted fever rickettsioses）是宿主感染立克次体属（Genus Rickettsia）革兰氏阴性专性胞内菌后的致病结局。尽管已有研究将长非编码RNA与感染及炎症通路相关联，但长非编码RNA在宿主对抗立克次体属病原菌的免疫应答中发挥的作用仍未明确。
本研究以可模拟人类斑点热立克次体病发病机制的柯氏立克次体（R. conorii）感染易感小鼠为模型，对感染过程中宿主长非编码RNA的表达谱进行了分析。对感染宿主肺部组织进行RNA测序后，共获得74964个匹配至非编码RNA的测序读段；与未感染对照组相比，其中206个非编码RNA显著上调，277个显著下调。
在剔除短非编码RNA与歧义转录本后，研究团队针对剩余转录本开展了深入分析：结合小鼠肺组织的表观遗传标记（组蛋白H3K4单甲基化（H3K4Me1）、组蛋白H3K4三甲基化（H3K4Me3））、活跃转录标记（POLR2A、p300、CTCF）以及DNaseI超敏位点，最终鉴定出两个具有潜在活性且显著上调的增强子长非编码RNA：NONMMUT013718与NONMMUT024103。
借助Hi-3C测序数据资源，研究团队进一步发现，NONMMUT013718与NONMMUT024103的基因组位点可分别与邻近的蛋白编码基因Id2（DNA结合抑制因子2，inhibitor of DNA binding 2）与Apol10b（载脂蛋白10b，apolipoprotein 10b）相互作用并调控其表达。
异源报告基因实验证实，这两种增强子长非编码RNA可诱导其预测靶标的蛋白编码基因表达。
感染小鼠的肺部组织中，这两种增强子长非编码RNA及其靶基因的表达水平均显著高于模拟感染对照组。体外实验中，柯氏立克次体感染小鼠巨噬细胞后可诱导NONMMUT013718与Id2的表达，感染内皮细胞后也可明显上调NONMMUT024103与Apol10b的表达。
最后，通过短发夹RNA（shRNA）介导敲低NONMMUT013718与NONMMUT024103的表达后，内源Id2与Apol10b的表达水平均出现下调，证实了这两种增强子长非编码RNA对其靶蛋白编码基因的调控作用。
本研究首次提供实验证据，表明在致病性立克次体感染过程中，肺部长非编码RNA的表达发生改变，且增强子长非编码RNA可调控免疫相关蛋白编码基因的表达。