Characterization of the essential chromatin remodeling enzyme pfSnf2L in Plasmodium falciparum

恶性疟原虫（Plasmodium falciparum, Pf）是一种单细胞真核生物，在人类宿主和蚊子媒介之间具有复杂的生命周期。每个生命周期阶段都有特定的转录谱，表明存在复杂的转录调控机制。该寄生虫是独一无二的，具有迄今为止最丰富的at基因组测序，真核生物区域中最多样化的组蛋白，导致缺乏全局核小体定位，并且与相似复杂性的生物体相比，转录因子数量减少了10倍。因此，目前尚不清楚发育阶段过渡过程中严格调控的基因表达谱是如何建立的。这表明基因表达级联的调控依赖于涉及核小体重塑的表观遗传机制。与其他真核生物不同，Pf具有一组非冗余的染色质重塑酶。这允许通过诱导敲除系统和相关功能研究来研究细胞中单个类别的染色质重塑者的功能。我们选择了iswi型酶pfSnf2L，建立了可诱导敲除的寄生虫系，结果表明pfSnf2L是寄生虫生存所必需的酶。pfSnf2L的条件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，在红细胞周期的正确阶段，输出组基因不会被激活，这与这种表型很好地吻合。该条件突变体将使我们能够解决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，并结合重组pfSnf2L酶的功能表征，绘制细胞染色质变化的机制图。我们旨在揭示恶性疟原虫染色质重塑和核小体定位对基因表达网络和表型变化的影响机制。

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.