Unravelling molecular mechanisms of transcriptional regulation by Plasmodium falciparum bromodomain proteins

疟疾寄生虫恶性疟原虫感染并在人类红细胞内繁殖。像所有活着的生物体一样，疟疾寄生虫需要严格调控自己的基因，以产生对生理过程至关重要的蛋白质，例如在正确的时间点入侵宿主细胞。基因调控涉及由动态结构和组成的染色质介导的表观遗传机制，染色质由包裹在组蛋白八聚体周围的重复DNA单元组成。组蛋白的N端末端可以用小的化学基团可逆地修饰，为各种蛋白质复合体创建一个高度动态的对接平台，可以改变染色质的结构，以时间和空间的方式控制基因的表达。一个重要的修饰是在组蛋白的赖氨酸残基上增加了一个乙酰基，它可以被含有称为溴域的模块的蛋白质结合。由于其在基因调控中的核心作用和已开发出的有效抑制剂的事实，溴结构域蛋白最近已成为艾滋病毒、癌症和炎症的局部药物靶点，并有望成为新型抗疟疾药物的靶点。我们最近发现了一种独特的恶性疟原虫溴结构域蛋白PfBDP1，它是一种组蛋白乙酰赖氨酸结合蛋白，对寄生虫的生长至关重要。PfBDP1与红细胞侵袭相关基因启动子中的组蛋白结合并协调其表达。然而，PfBDP1控制转录的分子机制尚不清楚。其他生物中的溴域蛋白通过与特定的转录因子和介体复合体相互作用来调节基因的表达，介体复合体是一种灵活的多亚基复合体，对几个步骤至关重要。这些相互作用导致染色质的局部变化，并最终导致RNA聚合酶II的转录。我们发现了PfBDP1在疟疾寄生虫中可能以类似的方式发挥作用的证据。因此，我们的目的是验证恶性疟原虫溴结构域蛋白PfBDP1通过与特定的转录因子和基本转录机制协同作用来控制转录的假设。我们将使用新兴的基因组编辑工具，并使用全基因组RNA和染色质测序(ChIPseq)以及蛋白质组学方法来揭示疟疾寄生虫中PfBDP1介导的基因调控的基本分子机制。更具体地说，我们将询问PfBDP1与ApiAP2转录因子家族的候选共激活因子(AIM1)的功能关系，我们将检测PfBDP1缺失对基础转录机制组件的影响(AIM 2)，并将从功能上表征与PfBDP1相互作用的新的染色质蛋白(AIM 3)。总之，这些研究将有助于更好地理解疟疾寄生虫是如何调控基因的，这些基因使它们能够在人类宿主中感染和生存。

The malaria parasite Plasmodium falciparum infects and multiplies inside human red blood cells. Like all living organisms, malaria parasites need to tightly regulate their genes to produce proteins important for physiological processes such as the invasion of their host cells at the correct point in time. Gene regulation involves epigenetic mechanisms mediated by the dynamic structure and composition chromatin, which consist of repetitive units of DNA wrapping around an octamer of histone proteins. The N-terminal tails of histones can be reversibly modified with small chemical moieties to create a highly dynamic platform of docking stations for various protein complexes that can modify the structure of chromatin and control gene expression in a temporal and spatial fashion. One important modification is the addition of an acetyl-group to lysine residues in histones, which can be bound by proteins that contain a module called a bromodomain. Due to their central role in gene regulation and the fact that potent inhibitors have been developed, bromodomain proteins have recently become topical drug targets in HIV, cancer, and inflammation and could be promising as targets for novel anti-malarials. We have recently identified a unique P. falciparum bromodomain protein, PfBDP1, as a histone acetyl-lysine binding protein that is essential for parasite growth. PfBDP1 binds to histones in the promoter of genes involved in erythrocyte invasion and coordinates their expression. However, the molecular mechanisms by which PfBDP1 controls transcription are unclear. Bromodomain proteins in other organisms regulate gene expression through interactions with specific transcription factors and the Mediator complex, a flexible multi-subunit complex critical for several steps. These interactions induce local changes in chromatin and ultimately lead to transcription by RNA Polymerase II. We have found evidence that PfBDP1 may act in a similar way in malaria parasites. Therefore we aim to test the hypothesis that the P. falciparum bromodomain protein PfBDP1 controls transcription by co-operating with specific transcription factors and the basal transcription machinery. We will use emerging genome-editing tools and employ genome-wide RNA- and Chromatin-sequencing (ChIPseq) as well as proteomics approaches to uncover fundamental molecular mechanisms of gene regulation mediated by PfBDP1 in the malaria parasite. More specifically, we will interrogate the functional relationship of PfBDP1 with a candidate co-activator of the ApiAP2 transcription factor family (aim1), we will examine the effect of PfBDP1 depletion on components of the basal transcription machinery (aim 2), and we will functionally characterize a putative novel chromatin protein that interacts with PfBDP1 (aim 3). Together, these studies will contribute to a much improved understanding of how malaria parasites regulate the genes that allow them to infect and survive in their human host.