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

对疟疾寄生虫传播重要的潜在表观遗传调节锌指蛋白的功能表征

• 批准号: 407527076
• 负责人: Dr. Che Julius Ngwa, Ph.D.
• 金额: --
• 依托单位: Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/407527076?language=en
• 关键词: Functional; characterization; potential; epigenetically; regulated

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.

在过去的十年中，基因调控的表观遗传机制因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，包括原生动物寄生虫，如恶性疟原虫，致命的热带疟疾的病原体。虽然表观遗传控制机制在恶性疟原虫的无性血液阶段已被广泛研究，并且被证明对于免疫逃避特别重要，但人们对配子细胞中的这些机制知之甚少。这些传染阶段在人类红细胞中发育 10 天，一旦被吸血按蚊吸收，就会进行配子发生以启动有性生殖。因此，配子体对于疟疾寄生虫从人到蚊子的传播起着至关重要的作用。为了更深入地了解配子细胞表观遗传基因调控机制，我们最近采用了一种使用组蛋白脱乙酰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过度乙酰化，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，我们鉴定了一小群编码锌指蛋白（ZFP）的基因，这些基因在 TSA 处理的配子体中转录水平高度上调，表明它们的表达水平可能受到组蛋白乙酰化的控制。 ZFP 是锌离子结合蛋白的一个多样化家族，可作为 DNA、RNA 和蛋白质的相互作用物，并且除其他外还充当转录因子。尽管恶性疟原虫基因组编码了 200 多种具有锌指结构域的蛋白质，但迄今为止，人们对它们在疟原虫生命周期中的作用知之甚少。为了获得恶性疟原虫配子细胞中七个ZFP功能的初步证据，在本研究中，我们旨在回答以下问题：1）配子体发育过程中七个ZFP参与哪些调控过程？ 2）七个ZFP的交互伙伴是哪些？ 3) 哪些基因组的表达水平会受到七个 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，例如基因敲除、基于 CRISPR/Cas 的基因编辑和 RNA 测序。因为在其他真核生物中，ZFP 代表了有希望的药物靶点，这些配子体特异性 ZFP 的详细功能表征可能有助于识别抗疟药的潜在靶点。