A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) [ChIP-Seq TF]

Gametocytogenesis, the process by which malaria parasites produce sexual forms that can infect mosquitoes, is essential for the transmission of malaria. A transcriptional switch of the pfap2-g gene triggers sexual commitment, but how the complex multi-step process is precisely programed remains largely unknown. Here, by systematic functional screening of a panel of ApiAP2 transcription factors, we identify six new apiAP2 members associated with gametocytogenesis in Plasmodium falciparum. Among these, PfAP2-G5 (PF3D7_1139300) was found to be indispensable for gametocytogenesis. This factor suppresses the transcriptional activity of the pfap2-g gene via binding to both the upstream region and exonic gene body, the latter is linked to the maintenance of local heterochromatin structure, thereby preventing initiation of sexual commitment. Removal of this repressive effect through pfap2-g5 knockout disrupts the asexual replication cycle and promotes sexual commitment accompanied by upregulation of pfap2-g expression. However, the gametocytes produced fail to mature fully. Further analyses show that PfAP2-G5 is essential for gametocyte maturation, and causes the down-regulation of pfap2-g and a set of early gametocyte genes activated by PfAP2-G prior to gametocyte development. Collectively, our findings reveal a regulation cascade of gametocyte production in malaria parasites, and provide a new target for transmission blocking interventions. The PfAP2-G5-GFP::WT line at trophozoite (30-40hpi) and G0-G1 gametocyte (D5) stages, the PfAP2-G-GFP::WT and PfAP2-G-GFP::pfap2-g5_trun lines at trophozoite (30-40hpi) stage were harvested to perform ChIP assay using antibody against GFP or IgG. There are two biological replicates for each of the parasite lines. The input and IgG material served as the control.

配子体发生（Gametocytogenesis）是疟原虫生成可感染蚊子的有性生殖形态的过程，对疟疾的传播具有关键意义。pfap2-g基因的转录开关可触发疟原虫的有性分化，但这一复杂多步骤过程的精准调控机制目前仍不甚明确。本研究通过对一组ApiAP2转录因子（ApiAP2 transcription factors）开展系统性功能筛选，在恶性疟原虫（Plasmodium falciparum）中鉴定出6个与配子体发生相关的新型ApiAP2家族成员。其中，PfAP2-G5（PF3D7_1139300）被证实为配子体发生所必需的因子。该因子通过结合pfap2-g基因的上游区域与外显子基因体，抑制其转录活性；其中结合外显子区域的过程与局部异染色质结构的维持相关，进而阻断有性分化的启动。通过敲除pfap2-g5解除该抑制效应后，会破坏疟原虫的无性繁殖周期，并促进有性分化，伴随pfap2-g表达水平上调。但所产生的配子体无法完全成熟。进一步分析显示，PfAP2-G5对配子体成熟同样不可或缺，其可下调pfap2-g以及一组由PfAP2-G在配子体发育前激活的早期配子体基因的表达。综上，本研究揭示了疟原虫配子体生成的调控级联通路，为疟疾传播阻断干预策略提供了全新靶点。本研究收集了处于滋养体期（30-40hpi）及G0-G1期配子体（第5天）的PfAP2-G5-GFP::WT虫株，以及处于滋养体期（30-40hpi）的PfAP2-G-GFP::WT与PfAP2-G-GFP::pfap2-g5_trun虫株，采用抗GFP抗体或IgG进行染色质免疫共沉淀（ChIP assay）实验。每个虫株均设置2个生物学重复，以Input样本及IgG样本作为对照。