Characterization of the essential chromatin remodeling enzyme pfSnf2L in Plasmodium falciparum

恶性疟原虫(Pf)是一种单细胞真核生物，在人类宿主和蚊媒之间有着复杂的生命周期。每个生命周期阶段都可以通过特定的转录图谱来表征，这意味着一个复杂的转录调控机制。这种寄生虫是独一无二的，具有迄今为止最丰富的AT基因组序列，是真核生物领域中分歧最大的组蛋白蛋白，导致缺乏全球核小体定位，与类似复杂性的生物体相比，转录因子的数量减少了10倍。因此，目前还不清楚发育阶段转变过程中严格调控的基因表达谱是如何建立的。与其他真核生物不同，PF具有一组非冗余的染色质重塑酶。这使得可以通过可诱导的基因敲除系统和相关的功能研究来研究细胞中个别类别的染色质重构体的功能。我们选择了ISWI型酶pfSnf2L，建立了可诱导的基因敲除寄生虫株系，表明pfSnf2L是寄生虫生存所必需的酶。PfSnf2L的条件性缺失导致寄生虫无法离开红细胞。基因表达分析表明，在缺乏pfSnf2L的情况下，出口体基因在红细胞内周期的正确阶段不会被激活，这与这种表型很好地一致。这个条件突变将使我们能够解决pfSnf2L在PF红细胞生命周期中对染色质结构和基因调控的影响。本研究结合重组pfSnf2L酶的功能特性，绘制了细胞染色质变化的机制图。我们旨在揭示染色质重塑和核小体定位对恶性疟原虫基因表达网络和表型变化的作用机制。

The malaria causing parasite Plasmodium falciparum (Pf) is an unicellular eukaryote with a complex life cycle between human host and mosquito vector. Each life cycle stage can be characterized by a specific transcriptional profile, suggesting a complex transcriptional regulation mechanism. The parasite is unique, having the AT-richest genome sequenced to date, the most divergent histone proteins in the eukaryotic domain, resulting in a lack of global nucleosome positioning and exhibiting a 10-fold reduction in number of transcription factors, when compared to organisms of similar complexity. Therefore, it is not understood how the tightly regulated gene expression profile during developmental stage transition is established. It is suggested that the regulation of the gene expression cascade depends on epigenetic mechanisms involving nucleosome remodeling.In contrast to other eukaryotes, Pf has a non-redundant set of chromatin remodeling enzymes. This allows the study of the function of individual classes of chromatin remodelers in the cell by inducible knockout systems and associated functional studies. We chose the ISWI-type enzyme pfSnf2L, created inducible knockout parasite lines, and show that pfSnf2L is an essential enzyme for parasite survival. Conditional deletion of pfSnf2L resulted in parasites unable to exit the erythrocyte. In good agreement with this phenotype, gene expression analysis demonstrated that exportome genes are not activated at the correct stage of the intraerythrocytic cycle, in the absence of pfSnf2L. This conditional mutant will allow us to address the effects of pfSnf2L on chromatin structure and gene regulation during the erythrocyte life cycle of Pf. The study is combined with the functional characterization of the recombinant pfSnf2L enzyme, to draw a mechanistic picture of the cellular chromatin changes. We aim to unravel the mechanism of chromatin remodeling and nucleosome positioning on gene expression networks and phenotypical changes of Plasmodium falciparum.