调控IFN-β转录的长链非编码RNA的发现与机制研究

Host defense against virus invasion is initiated through recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) and resulted in the activation of IFN transcription. LncRNAs have provided a new perspective on gene regulation in diverse biological contexts. Systematic detection of functional lncRNAs and their biological contexts is required to understand the mechanisms by which variation in lncRNAs expression contributes to the development and activation of immune cells. Functional lncRNAs can be detected by lncRNA expression profiling and protein-binding RNA profiling, but these approaches cannot detect lncRNAs that directly regulate transcription of a particular gene. We hypothesized that recruitment of lncRNAs to promoter region would be an essential prerequisite of transcription modulation, even if the lncRNA is not directly binding to promoter DNA. Here we plan to build-up a novel platform named “Promoter-IP” , to specific immunoprecipitated the entire gene promoter, as well as any binding lncRNA. Therefore, the promoter-associated lncRNAs that can be identify by high-throughput RNA sequencing. By applying the “Promoter-IP” system to Ifn-β gene, we could identify functional lncRNAs that might play an important role in Ifn-β production from the “promoter-region binding lncRNA profile”. And further study would elucidate the mechanisms underlying the lncRNA`s transcriptional regulatory function to Ifn-β transcription. This study would gain us further understanding to the Ifn-β transcription regulation and attribute to the discovery of novel therapy to viral infection related diseases.

IFN-β在机体抵抗病毒免疫过程中发挥着至关重要的作用，其转录过程，主要由位于启动子区的转录增强复合物（enhancesome）所调控。长链非编码RNA（lncRNA）是近些年发现的具有丰富调控功能的RNA分子。lncRNA不编码蛋白，在细胞生命活动过程中，特别是在基因转录过程中，可以通过多种机制，结合多种蛋白质分子或基因组DNA序列，发挥调控功能。然而，lncRNA分子对IFN-β转录的调控，目前的研究还处于空白。为了研究调控IFN-β 转录的lncRNA，本项目拟结合CRISRP/Cas9系统、免疫共沉淀和高通量测序检测，建立启动子免疫共沉淀体系（Promoter IP）。通过运用Promoter-IP，检测IFN-β启动子区结合的lncRNA，从中找到发挥关键调控功能的lncRNA分子。进而通过深入的机制研究，发现lncRNA调控IFN-β转录的新机制，为感染性疾病的治疗提供新思路。

本项目按照申请书中的研究计划执行，通过建立并优化Promoter-IP体系，发现了调控IFN-β转录的关键lncRNA分子——Neat1，并深入研究了Neat1调控转录的分子机制，找到了抗病毒免疫应答调控的新机制。在病毒感染的早期，Ⅰ型干扰素表达的强弱，对于感染性疾病的发展、病程和转归具有关键作用。本项目通过建立基于CRISPR技术的目标基因启动子区靶向结合的lncRNA检测技术——Promoter-IP技术，绘制了结合在IFN-β启动子区的lncRNA图谱，并从中筛选出一个可以结IFN-β启动子区的lncRNA：Neat1。功能学研究发现，在小鼠成纤维细胞中敲除Neat1基因后，严重影响了IFN-β基因的正常转录，并且病毒在细胞内的复制加剧。进一步分析细胞信号转导网络发现，Neat1可以增强细胞识别、启动IFN-β转录的信号转导、提高关键转录因子IRF3和IRF7的磷酸化水平。由于IRF3、IRF7与Neat1的细胞亚定位不同，Neat1并不是直接调控转录因子的磷酸化或去磷酸化水平，而是通过调节转录因子在细胞核内的滞留来发挥功能。以往研究发现，Neat1可以作为分子骨架，在细胞核内形成核旁斑（Paraspeckle）结构，特异性的与基因组中转录活化的区域相结合。我们发现Neat1可以在IFN-β的启动子区组装Paraspeckle，干扰参与Paraspeckle结构组装的蛋白的表达，细胞出现与Neat1敲除类似IFN-β转录被抑制的现象。Paraspeckle调控IFN-β转录的具体机制，是将多个转录活化的区域在空间上相互聚集，形成转录热点。而IFN-β的转录热点还存在“耦合增强”功能：即当细胞同时受到病毒刺激和IFN-β刺激时，可以进一步增强IFN-β的转录水平。Neat1形成的Paraspeckle还可以作为“炎症消退感受器”，在感知到适应性免疫应答激活，巨噬细胞活化后分泌的代谢产物25-羟基胆甾醇后，会解除这种耦合增强功能，促进炎症的消退和病毒的清除。