恶性疟疾致病相关var基因非编码核酸序列的表达调控及机制研究

Plasmodium falciparum (Pf) is the major pathogen of malaria, which causes more than one million deaths of infected peoples worldwide. The Pf variant gene family (Pfvar) is the critical virulence factor responsible for the pathogenesis in severe malaria. There are approximately 60 members in var gene family, but only one will be expressed in each intraerythrocytic cycle while the others are silencing. This "mutually exclusive expression" system had been shown to be regulated by epigenetic factors including perinuclear localization, re-location between active and silent zones, and chromatin modification in var promoter region. Recently, we revealed for the first time that the interaction between a highly conserved intronic motif (iNPE) and nuclear actin played a crucial role in perinuclear anchoring and repositioning of var genes, indicating the non-coding sequences are one of the main regulators of var gene expression. However, the intron/actin complex is likely only regulating the B and C-subtype var genes, and the detailed mechanism is still not completely elucidated. On the basis of our novel finding, this study aims to identify the regulatory motif in all of the non-coding sequences, i.e. 5'UTR, intron, and 3'UTR, their transcripts, and the non-coding DNA/RNA binding proteins for the A-subtype var genes, which has been demonstrated to be associated with lethal malarial cases, and elucidate the related pathways and mechanisms by strategies including transfection, Knock out, Knock down, chromatin immunoprecipitation, identification of DNA/RNA-binding proteins, and high resolution RNA sequencing, etc. The accomplishment of this project would definitely provide new targets and tools for prevention and therapy of the severe malaria.

目前全球每年仍有一百万以上人死于疟疾，主要是由恶性疟原虫（Pf）感染所致。var变异基因家族是Pf致病相关的关键毒力因子，含约60个成员，可分为不同的亚类。 疟原虫每个红内期裂体增殖周期只表达其中一个var基因，其余均沉默。Pfvar基因这种"排他性"表达机制与其核周锚定、迁移、染色质修饰等表观遗传学调控有关。申请人在前期研究中，已发现var基因内含子保守序列（iNPE）与核周肌动蛋白的相互作用介导var基因核周定位与迁移。然而，此机制只涉及B/C亚类的var基因转录调控。本课题将在此基础上，采用转基因和基因敲除技术、核酸结合蛋白质鉴定技术、染色质免疫沉淀、以及RNA深度测序等技术，对强毒力相关的A亚类var基因相关非编码区及其转录产物的关键调控序列及其结合蛋白开展系统的鉴定和功能分析，阐明其作用机制。本课题将为阐明var基因的表达调控机制并为致死型重症疟疾的干预措施研制开创机遇。

恶性疟原虫感染是引起疟疾死亡的主要原因，每年造成约一百万人死亡，其“首要毒力因子”是恶性疟原虫变异基因家族var基因编码的多功能大分子蛋白-恶性疟原虫红细胞膜蛋白1（PfEMP1）。恶性疟原虫含有60个var基因，但在一个红细胞裂体增殖周期中一般只能优势表达其中一个，这种“排他性表达”受到诸多因素的调控，包括染色质修饰、染色质环境改变及染色质重构等转录前调控过程，但具体的调控机制尚不完全清楚。本项目在前期工作基础上，利用双荧光素报告基因分析、核酸结合蛋白分离与鉴定技术、HA表位标记或GFP转基因技术、间接免疫荧光/荧光原位杂交技术(IFA/FISH)、染色质免疫沉淀技术（ChIP）、紫外交联染色质免疫沉淀（HITS-CLIP)等方法，针对恶性疟原虫毒力var基因相关非编码区的关键调控序列（以内含子为重点，兼顾上、下游非编码区序列）、ncDNA/ncRNA结合蛋白进行系统地鉴定。本课题发现var基因下游非编码核酸序列GC-elements对侧翼var基因的转录沉默具有顺式调控作用，很好地阐明了染色质边界隔离（元件）机制。 此外，var基因内含子结合蛋白PFE0435cc，PF10_0068和PFI1435w在体外可与内含子保守序列iNPE存在特异结合，且PFE0435cc除了与iNPE发生结合，有可能介导了PF11_0091与其他元件相互作用参与var基因的抗原变异调控。这些对多因素共同参与的var基因转录调控协调机制进行了更为合理的阐释。本课题结果还表明var基因启动子结合的染色质重构蛋白PF11_0053 和PFF0560c可能参与核周区域C亚类var基因的转录调控，PFF0560c蛋白敲除后影响了恶性疟原虫的正常发育和生长，初步阐明了该类染色质重构因子调控疟原虫毒力基因克隆变异的分子机制。这些成果为全面系统揭示恶性疟原虫var基因独特的表达机制，继而研发相应的干预措施以降低疟疾死亡率提供基础。