Functional characterization of potential epigenetically regulated zinc finger proteins important for malaria parasite transmission

在过去的十年中，基因调控的表观遗传机制由于其在细胞分化和细胞周期控制中的关键作用而受到越来越多的关注。严格控制基因调控对于具有高复制率的细胞尤其重要，包括恶性疟原虫等原生动物寄生虫，恶性疟原虫是致命的热带疟疾的病原体。虽然表观遗传控制机制已在恶性疟原虫的无性血液阶段进行了广泛的研究，并在这里被证明是特别重要的免疫逃避，很少有人知道这些机制在配子体。这些传播阶段在10天的时间内在人类红细胞中发育，一旦被吸血按蚊摄取，就会进行配子发生以启动有性生殖。因此，配子细胞在疟疾寄生虫的人-蚊传播中起着至关重要的作用。为了更深入地了解配子体中表观遗传基因调控的机制，我们最近采用了一种使用组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）的化学功能丧失技术。TSA处理损害配子体成熟并导致组蛋白超乙酰化，从而导致不同配子体阶段超过294个基因的转录失调。有趣的是，我们确定了一个小组的7个基因编码的锌指蛋白（ZFP），这是高度上调的转录水平在TSA处理的配子母细胞，表明它们可能是控制在其表达水平的组蛋白乙酰化。ZFP是锌离子结合蛋白的不同家族，其充当DNA、RNA和蛋白质的相互作用物，并且其中充当转录因子。虽然恶性疟原虫基因组编码超过200种具有锌指结构域的蛋白质，但迄今为止对它们在疟原虫生命周期中的作用知之甚少。为了获得这7种ZFP在恶性疟原虫配子母细胞中功能的初步证据，本研究旨在回答以下问题：1）这7种ZFP在配子母细胞发育过程中参与哪些调控过程？2)7个ZFP的互动伙伴是哪些？3)哪些基因组的表达水平会受到这七种ZFP的影响？为了回答这些问题，我们将采用免疫组织化学方法和分子方法，如基因敲除，基于CRISPR/Cas的基因编辑和RNA测序。因为在其他真核生物中，ZFP代表有前途的药物靶点，这些配子体特异性ZFP的详细功能表征可能导致抗疟药的潜在靶点的鉴定。

During the last decade, epigenetic mechanisms of gene regulation have gained increasing attention due to their crucial roles in cellular differentiation and cell cycle control. A tight control of gene regulation is particularly important for cells with high replication rates including protozoan parasites like Plasmodium falciparum, the causative agent of the deadly malaria tropica. While epigenetic control mechanisms have been studied extensively in the asexual blood stages of P. falciparum and were here shown to be particularly important for immune evasion, little is known about these mechanisms in gametocytes. These transmissible stages develop in the human red blood cells during a period of 10 days and once being taken up by a blood-feeding Anopheles mosquito undergo gametogenesis to initiate sexual reproduction. Gametocytes thus have crucial for roles for human-to-mosquito transmission of the malaria parasite. To gain more insights into the mechanisms of epigenetic gene regulation in gametocytes, we recently employed a chemical loss-of-function technique using the histone deacetylase inhibitor Trichostatin A (TSA). TSA-treatment impaired gametocyte maturation and lead to histone hyper-acetylation, thereby resulting in the transcriptional deregulation of over 294 genes in the different gametocyte stages. Interestingly, we identified a small group of seven genes encoding for Zinc finger proteins (ZFPs), which were highly upregulated in their transcript levels in TSA-treated gametocytes, indicating that they may be controlled in their expression levels by histone acetylation. ZFPs are a diverse family of zinc ion-binding proteins that serve as interactors for DNAs, RNAs, and proteins and which among others function as transcription factors. Although the P. falciparum genome encodes more than 200 proteins with zinc finger domains, to date not much is known about their roles during the life cycle of the malaria parasite. In order to gain initial evidence for the function of the seven ZFPs in the P. falciparum gametocytes, in this study we aim to answer the following questions: 1) In which regulatory processes are the seven ZFPs involved during gametocyte development? 2) Which are the interaction partners of the seven ZFPs? 3) Which gene groups would be affected in their expression levels by the seven ZFPs? To answer these questions, we will employ methods of immunohistochemistry with molecular approaches like gene-knock down, CRISPR/Cas-based gene editing and RNA sequencing. Because in other eukaryotes, ZFPs represent promising drug targets, the detailed functional characterization of these gametocyte-specific ZFPs may lead to the identification of potential targets for antimalarials.