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体红细胞内繁殖。像所有生物一样，疟原虫需要严格调节它们的基因，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。基因调控涉及由染色质的动态结构和组成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DNA重复单元组成。组蛋白的N-末端尾部可以用小的化学部分可逆地修饰，以产生用于各种蛋白质复合物的对接站的高度动态平台，所述蛋白质复合物可以以时间和空间方式修饰染色质的结构并控制基因表达。一个重要的修饰是在组蛋白中的赖氨酸残基上添加乙酰基，该组蛋白可以被包含称为溴结构域的模块的蛋白质结合。由于它们在基因调控中的核心作用以及已经开发出有效抑制剂的事实，溴结构域蛋白最近已成为HIV、癌症和炎症中的局部药物靶标，并且可能有希望作为新型抗疟疾药物的靶标。我们最近鉴定了一种独特的恶性疟原虫布罗莫结构域蛋白PfBDP 1，作为寄生虫生长所必需的组蛋白乙酰赖氨酸结合蛋白。PfBDP 1与参与红细胞侵袭的基因启动子中的组蛋白结合并协调其表达。然而，PfBDP 1控制转录的分子机制尚不清楚。其他生物体中的溴结构域蛋白通过与特异性转录因子和介体复合物（一种对几个步骤至关重要的灵活多亚基复合物）相互作用来调节基因表达。这些相互作用诱导染色质的局部变化，并最终导致RNA聚合酶II的转录。 我们已经发现证据表明PfBDP 1可能以类似的方式在疟疾寄生虫中起作用。因此，我们的目标是测试恶性疟原虫布罗莫结构域蛋白PfBDP 1通过与特异性转录因子和基础转录机制合作来控制转录的假设。我们将使用新兴的基因组编辑工具，并采用全基因组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PfBDP 1介导的基因调控的基本分子机制。更具体地说，我们将询问PfBDP 1与ApiAP 2转录因子家族（aim 1）的候选共激活因子的功能关系，我们将检查PfBDP 1缺失对基础转录机制组分的影响（aim 2），我们将功能性地表征与PfBDP 1相互作用的推定的新染色质蛋白（aim 3）。总之，这些研究将有助于更好地了解疟原虫如何调节基因，使它们能够感染并在人类宿主中生存。

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.