Chromatin structure and control of gene expression in the human malaria parasite

摘要： 世界上近一半的人口生活在疟疾流行的国家。疟原虫 恶性疟原虫是人类最严重的疟疾形式的病原体， 造成全球95%的疟疾死亡。该项目的主要目标是 描述控制染色质组织和基因的分子决定因素 对这种病原体的调节；阐明它们在寄生虫发育、毒力、 和性别分化；并找出可以靶向杀死 寄生虫。这项拟议的研究建立在大量分子、细胞和 在pl的实验室中产生的全基因组数据发现了3D 基因组组织和转录调节性分化和寄生虫的发育。 尽管在过去的10年里在阐明染色质的作用方面取得了重大进展 转录调控、染色质结构改变的机制及其影响因素 控制这些变化的机制仍有待阐明。这里提出的研究将检验 参与染色质调控的蛋白质和长非编码RNA(LncRNA) 结构、寄生虫发育、毒力和性别分化。该项目是 组织成三个具体目标。目标1将验证以下内容的作用和重要性 我们之前确定的两种蛋白质，它们可能是男性的主要调节因子 和女性的性别分化。编码AP2转录因子的两个基因 (PfApi2AP2-Fg)和丝氨酸/苏氨酸蛋白磷酸酶2A激活剂(PfPTPA) 位于两个染色质超密集结构域的边界 早配子体和晚配子体。我们将全面研究这些蛋白质的功能。 在雄配子体和雌配子体发育过程中使用细胞、分子和遗传学 接近了。AIM 2将验证两个lncRNAs(lncRNAG9和lncRNAG14)的作用 以前被认为是性分化和染色体的潜在调节者 在寄生虫发育过程中的重组。这些因素可能与染色体有关。 配子体阶段的重组。因此，我们将实现一组蜂窝、 分子和遗传学方法，以表征它们在细胞的形成和维持中的作用 寄生虫性阶段，以及我们观察到的特定的染色体重组 这些阶段。目标3将系统地分离和鉴定蛋白质和lncRNAs 控制转录沉默的异染色质的结构和活性 使用一种名为染色质分离的新方法在恶性疟原虫基因组中聚集 经RNA纯化(ChlRP)。一旦确定，因素将在 使用细胞和分子实验方法，包括基因组的功能水平 编辑：CRlSPR-Cas9。预计拟议的研究将提供 对寄生虫特异性蛋白复合体和lncRNAs及其基因的开创性新见解 在染色质结构和寄生虫生物学中的作用，以及小说的许多起点 疟疾研究和治疗方面的指导。

Abstract: Nearly half of the world's population lives in countries where malaria is endemic. Plasmodium falciparum, the causative agent of the most severe form of human malaria, is responsible for 95% of malaria deaths worldwide. This project's main goals are to characterize the molecular determinants that control chromatin organization and gene regulation in this pathogen; elucidate their importance in parasite development, virulence, and sexual differentiation; and identify novel pathways that can be targeted to kill the parasite. The proposed research builds upon a large body of molecular, cellular, and genome-wide data generated in the Pl's lab that discovered how close interconnections between 3D genome organization and transcription regulate sexual differentiation and parasite development. Despite significant progress in the past 10 years in elucidating the role of chromatin in transcriptional control, the mechanism underlying changes in chromatin structure and the factors controlling these changes remain to be elucidated. The studies proposed here will examine the proteins and long non-coding RNAs (lncRNAs) involved in the control of chromatin structure, parasite development, virulence, and sexual differentiation. The project is organized into three Specific Aims. Aim 1 will validate the role and essentiality of two proteins that we previously identified as potential master regulators of male and female sexual differentiation. Two genes encoding an AP2 transcription factor (PfApi2AP2-FG) and a serine/threonine protein phosphatase 2A activator (PfPTPA) are located at the boundary of two condensed chromatin super domains observed in both early and late gametocytes. We will fully investigate the function of these proteins during male and female gametocyte development using cellular, molecular, and genetic approaches. Aim 2 will validate the role of two lncRNAs (lncRNAG9 and lncRNAG14) that we previously identified as potential regulators of sexual differentiation and chromosome reorganization during parasite development. These factors may be involved in chromosome reorganization in gametocyte stages. We will therefore implement a set of cellular, molecular, and genetic approaches to characterize their role in the formation and maintenance of parasite sexual stages, as well as in the specific chromosomal reorganization we observed at these stages. Aim 3 will systematically isolate and identify the proteins and lncRNAs that control the structure and activity of transcriptionally silent heterochromatin clusters in P. falciparum genome, using a novel methodology called chromatin isolation by RNA purification (ChlRP). Once identified, factors will be validated at the functional level using cellular and molecular experimental approaches, including genome editing by CRlSPR-Cas9. lt is anticipated that the proposed research will offer groundbreaking new insights into parasite-specific protein complexes and lncRNAs and their role in chromatin structure and parasite biology, as well as many starting points for novel directions in malaria research and therapy.