Histone methylation during Plasmodium falciparum sexual differentiation

2002年，人类恶性疟原虫基因组的测序为发现新的药物和疫苗靶点带来了希望。在接下来的几年里，转录组学和蛋白质组学帮助将这些基因分配给不同的寄生虫阶段，并允许第一次瞥见生命周期进展所需的严格调控的转录程序。从那时起，越来越多的证据表明表观遗传学对疟原虫基因调控的重要贡献，特别强调调节红细胞复制阶段的组蛋白翻译后修饰。然而，当寄生虫需要准备从人类传播到蚊子时，组蛋白修饰扮演的角色还没有很好的理解。负责寄生虫传播的是配子体，配子体在人类红细胞中进行性分化，一旦被吸血按蚊摄取，就进入配子发生以启动有性生殖。在配子母细胞发育和随后的配子发生过程中，不同的基因库以协调一致的顺序打开和关闭，这表明基因表达受到严格的调控。我们最近证明了组蛋白修饰在发育中的配子体转录控制过程中的重要作用。虽然组蛋白去乙酰化沉默与无性血液和蚊子特异性阶段相关的基因，但组蛋白乙酰化激活对配子发生重要的基因。此外，赖氨酸特异性组蛋白甲基转移酶的化学抑制指向疟原虫SET结构域蛋白在配子体诱导和配子发生过程中的关键作用。然而，SET蛋白在配子体发育中的确切功能尚不清楚。因此，研究恶性疟原虫性分化过程中组蛋白甲基化的转录调控机制是本研究的首要目标。为了实现这一目标，我们将通过基因敲除，然后进行比较转录组学，对配子细胞特异性组蛋白甲基转移酶SET 2和SET 10进行功能表征（目的1）。此外，SET 2和SET 10的相互作用将通过生物素邻近标记鉴定来揭示，而受SET 2和SET 10影响的染色体区域将通过染色质免疫沉淀来鉴定（目的2）。此外，还未充分研究的S-腺苷甲硫氨酸合成酶SAMS，甲基化反应的关键调节剂，在配子体的转录控制过程中的参与将进行分析，使用基因敲除，然后生物化学和RNA测序方法（目的3）。这项研究获得的数据将提供新的见解组蛋白甲基化过程中的疟疾寄生虫的性分化，并将促进评估表观遗传调节剂和效应器作为抗疟疾的目标。

Sequencing of the genome of the human malaria parasite Plasmodium falciparum in the year 2002 fueled the hope for the discovery of novel drug and vaccine targets. In the following years, transcriptomics and proteomics helped to assign these genes to the distinct parasite stages and allowed a first glimpse on the tightly regulated transcriptional programme required for life-cycle progression. Since then increasing evidence points to a strong contribution of epigenetics to gene regulation in Plasmodium with particular emphasis on histone post-translational modifications regulating the erythrocytic replication phase. It is not yet well understood, though, which role histone modifications play, when the parasites need to prepare for transmission from the human to the mosquito. Responsible for parasite transmission are the gametocytes, which sexually differentiate in human red blood cells and, once being taken up by a blood-feeding Anopheles mosquito, enter gametogenesis to initiate sexual reproduction. During gametocyte development and following gametogenesis, different repertoires of genes are switched on and off in a well-coordinated sequence, pointing to a strict regulation of gene expression. We recently demonstrated a crucial role of histone modifications during transcriptional control in the developing gametocytes. While histone deacetylation silences genes related to the asexual blood and mosquito-specific stages, histone acetylation activates genes important for gametogenesis. Additionally, chemical inhibition of lysine-specific histone methyltransferases points to a pivotal role of plasmodial SET-domain proteins during gametocyte induction and gametogenesis. The exact functions of the SET proteins for gametocyte development, however, are not yet known. It is thus the overarching goal of this proposal to investigate the mechanisms of transcriptional regulation by histone methylation during P. falciparum sexual differentiation. To follow this goal, we will functionally characterize the gametocyte-specific histone methyltransferases SET2 and SET10 via gene-knockout followed by comparative transcriptomics (aim 1). Further, interactors of SET2 and SET10 will be unveiled by biotin proximity labelling identification, while chromosome regions affected by SET2 and SET10 will be identified by chromatin immunoprecipitation (aim 2). Additionally, the involvement of the yet under-investigated S-adenosylmethionine synthetase SAMS, a key regulator of methylation reactions, during the transcriptional control in gametocytes will be analysed, using gene-knockdown followed by biochemical and RNA sequencing approaches (aim 3). Data gained by this study will provide novel insights into histone methylations during sexual differentiation of the malaria parasite and will promote the evaluation of epigenetic regulators and effectors as antimalarial targets.