染色质重构因子PfCRCact调控恶性疟原虫毒力基因表达的功能与机制研究

Malaria is still prevalent throughout the world, and Plasmodium falciparum is the most harmful Plasmodium species which can cause severe malaria. Plasmodium virulence genes, var genes, exhibit unique expression pattern, including “mutually exclusive expression” and “clonal variation”, therefore result in a high level of antigenic variation, immunity evasion and plasmodium falciparum pathopoiesis. This kind of expression patterns involves a complex regulatory network of epigenetic regulation including chromatin modification and remodeling. In previous study, we found that Actin mediates the regulation of var genes which located in perinuclear region of heterochromatin. Further study showed that Actin-related chromatin remodeling factor PfCRCact is involved in the regulating process of var genes and acts synergistically with Actin, leading to the chromatin remodeling on local var gene sites and clonal variation of var genes. Here we propose a new conditional knockout system to study the function and mechanism of chromatin remodeler PfCRCact in regulating var gene expression. Through RNA-sequencing, micro-array, growth curve analysis (GCA) and local chromatin remodeling assay, we analyze and describe the in vivo and in vitro function of PfCRCact in chromatin remodeling on local var gene. We also study the mechanism of PfCRCact in regulating var gene expression by using chromatin immunoprecipitation assay (ChIP), Co-immunoprecipitation (Co-IP), indirect immunoinfluscent assay (IFA) and fluorescence in situ hybridization (FISH). This program is expected to elucidate the mechanism and function of PfCRCact in regulating the clonal variation of var genes, and provide scientific basis for malaria control.

疟疾仍肆虐全球许多国家或地区，其中恶性疟疾造成的危害最大。恶性疟原虫毒力基因（var基因）因其独特的表达模式，形成恶性疟原虫高度的抗原变异和免疫逃避，导致恶性疟疾发生。这种表达模式涉及染色质修饰和变构等表观遗传调控。我们前期研究发现肌动蛋白Actin介导了恶性疟原虫核周异染色质区var基因的表达调控，该调控还需一类Actin相关的染色质重构因子PfCRCact来协同完成var基因局部染色质改变，产生克隆变异。因此本项目拟采用一种新型的条件性敲除系统对PfCRCact进行研究：借助芯片杂交、生长曲线测定和核小体重建等技术完成其参与基因局部染色质变构的体内和体外功能分析；采用染色质免疫共沉淀、蛋白质免疫共沉淀、间接免疫荧光和荧光原位杂交等技术探讨其调控var基因表达的作用机制。从而阐明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，为恶性疟疾防治提供有价值的理论依据。

恶性疟原虫中存在多种转录因子参与了毒力基因家族（var基因）的互斥表达、克隆变异以及转录调控过程。最近研究表明：恶性疟原虫染色质重构因子PfCRCact（PfSWIB, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin）可能触发阶段特异性的程序性细胞死亡（PCD），不仅对疟原虫的生长发育至关重要，而且可能通过与其他未知蛋白发生相互作用，从而对疟原虫产生影响；然则目前，PfSIWB是否参与毒力基因的转录调控，以及如何发挥作用尚不清楚。本研究将条件性敲除系统PfSWIB-FKBP-LID引入恶性疟原虫3D7标准株，通过BSD药物循环和单克隆筛选，获得整合型虫株PfSWIB-HA-FKBP-LID；在此基础上对其进行生长曲线分析（GCA），即通过计算不同时间点原虫率，来观察不同虫株（PfSWIB敲除株、PfSWIB对照株和3D7标准株）在体外培养96小时（2个生活史）的生长发育情况；其次，我们通过qPCR分析来比较恶性疟原虫单个var基因在不同虫株的单一生活史内滋养体/环状体表达值变化，来证实var基因互斥性表达模式的改变；此外，采用高通量测序（RNA-seq）方法对不同虫株的var基因表达谱进行分析。生长曲线分析结果表明：PfSWIB条件性敲除可影响疟原虫的生长发育过程，与PfSWIB对照株和3D7标准株相比，PfSWIB∆（PfSWIB敲除株）的原虫率在体外培养至96h时显著下降（P<0.0001）；qPCR分析和RNA测序证实PfSWIB敲除后不仅使upsA、upsC和部分upsB类var基因“沉默”，而且导致部分原先沉默的upsB类var基因发生转录“活化”；同时，引起了upsA和部分upsB/upsC类var基因在恶性疟原虫晚期阶段的异常表达，即滋养体时期的upsA和部分upsB/upsC类var基因表达下调模式被逆转。在本研究中，我们证明了肌动蛋白相关的染色质调控因子PfSWIB参与了var基因表达的克隆变异过程，且对恶性疟原虫的生长发育至关重要，这些发现将有助于阐明PfSWIB调控疟原虫毒力基因-var基因家族表达转换和克隆变异的机制和功能。