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

摘要： 世界上将近一半的人口生活在疟疾流行的国家。疟原虫 恶性疟原虫是最严重的人类疟疾的病原体， 占全球疟疾死亡人数的95%。该项目的主要目标是 表征控制染色质组织和基因的分子决定因素 调节这种病原体;阐明其在寄生虫发育，毒力， 和性别分化;并确定新的途径，可以有针对性地杀死 寄生虫拟议的研究建立在大量的分子，细胞和 在PI的实验室中产生的全基因组数据发现了3D之间的紧密联系， 基因组组织和转录调节性分化和寄生虫发育。 尽管在过去的10年里，在阐明染色质在细胞中的作用方面取得了重大进展， 转录控制，染色质结构变化的机制和因子 控制这些变化仍有待阐明。这里提出的研究将审查 参与染色质控制的蛋白质和长链非编码RNA（lncRNA） 结构、寄生虫发育、毒力和性别分化。该项目 分为三个具体目标。目标1将确认以下方面的作用和必要性： 这两种蛋白质是我们之前发现的男性生殖系统潜在的主要调节因子， 女性的性别分化。两个编码AP 2转录因子的基因 在一些实施方案中，本发明涉及丝氨酸/苏氨酸蛋白磷酸酶2A激活剂（PfApi 2AP 2-FG）和丝氨酸/苏氨酸蛋白磷酸酶2A激活剂（PfPTPA）。 位于两个浓缩的染色质超结构域的边界， 早期和晚期配子体。我们将充分研究这些蛋白质的功能 在雄性和雌性配子体发育过程中，使用细胞、分子和遗传 接近。目的2将验证两种lncRNA（lncRNAG 9和lncRNAG 14）的作用， 以前被认为是性分化和染色体的潜在调节因子 寄生虫发育过程中的重组。这些因素可能参与了染色体 配子体阶段的重组。因此，我们将实施一套蜂窝， 分子和遗传方法来表征它们在形成和维持 寄生虫的性阶段，以及在特定的染色体重组，我们观察到， 这些阶段。目的3系统地分离和鉴定蛋白质和lncRNA 控制转录沉默异染色质的结构和活性 利用一种称为染色质分离的新方法， 通过RNA纯化（ChlRP）。一旦确定，将在 功能水平使用细胞和分子实验方法，包括基因组 通过CRISPR-Cas9编辑。预计这项研究将提供 对寄生虫特异性蛋白复合物和lncRNA及其 在染色质结构和寄生虫生物学中的作用，以及许多新的起点 疟疾研究和治疗的方向。

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.