The role of the interaction between the C-terminal domain of RNA pol II and a his

描述（由申请人提供）：RNA pol II 的 C 末端结构域与组蛋白甲基转移酶之间的相互作用在介导疟疾毒力基因的表观遗传记忆中的作用。 恶性疟原​​虫是人类疟疾最急性和最严重形式的原因。这种原生动物寄生虫感染人类宿主的循环红细胞，将主要毒力和抗原决定簇（一种称为 PfEMP1 的粘附蛋白）置于受感染的细胞表面。不同形式的 PfEMP1 由大型多拷贝 var 基因家族的各个成员编码。一次仅表达一个 var 基因。改变 var 基因的表达有助于寄生虫逃避免疫系统清除的能力，这被称为抗原变异。人们对调节各个 var 基因表达的机制知之甚少，但最近的工作表明组蛋白修饰发挥着重要作用。一旦 var 基因被激活，它往往会在许多细胞分裂中保持活跃，这种特性被称为“表观遗传记忆”。研究还表明，RNA 聚合酶 II 的主动转录对于维持恶性疟原虫 var 基因的表观遗传记忆是必需的，这表明聚合酶本身可能在维持调节 var 基因表达所需的染色质修饰方面发挥作用。此外，最近的研究表明，啮齿动物寄生虫不利用表观遗传记忆来控制其毒力基因的表达。系统发育比较揭示了灵长类寄生虫 RNA pol II 最大亚基的 C 端结构域 (CTD) 的异常扩展，而在啮齿类动物、鸟类或蝙蝠的寄生虫中未发现这种情况。此外，RNA pol II 的这个区域已被证明与高等真核生物中的染色质修饰酶相互作用，表明它可能参与表观遗传记忆的维持。有趣的是，两种特定的组蛋白修饰蛋白，组蛋白甲基转移酶 PfSet2 及其同源去甲基化酶 PfJmjC1，同样只在灵长类寄生虫中发现。先前在酵母中的结构和生化研究表明，RNA pol II CTD 的磷酸化形式可以直接与 Set2 相互作用，将其募集到活跃转录的基因中，从而有助于在基因组的高度转录区域维持活跃的染色质。 我们的假设是，PfSet2 通过与恶性疟原虫 RNA pol II 的磷酸化 CTD 相互作用，被招募到转录活跃的 var 基因中，以增强表观遗传记忆。本研究的目的是利用生化和结构方法确定 PfSet2 是否与 CTD 直接相互作用，并通过在恶性疟原虫中引入 PfSet2 的突变形式来研究这种相互作用在维持 var 基因表观遗传记忆中的作用。长期目标是确定 CTD 和 PfSet2 之间的相互作用在促进恶性疟原虫抗原变异等毒力机制中的作用。 公共卫生相关性：每年，全世界有数百万人受到恶性疟的困扰，导致撒哈拉以南非洲地区约一百万人死亡，其中大部分是幼儿。先前的研究表明，在人类红细胞上放置一种名为 PfEMP1 的抗原决定簇对于恶性疟原虫的生存至关重要，并且受到 var 基因转换机制的调节，该机制有助于寄生虫逃避免疫系统的能力。了解这种基因转换机制的调节可以导致开发更有效的药物，甚至可能开发出针对这种急性和严重疾病的疫苗。

DESCRIPTION (provided by applicant): The role of the interaction between the C-terminal domain of RNA pol II and a histone methyltransferase in mediating epigenetic memory in malaria virulence genes Plasmodium falciparum is responsible for the most acute and severe form of human malaria. This protozoan parasite infects the circulating red blood cells of its human host, placing on the infected cell surface the primary virulence and antigenic determinant, an adhesive protein called PfEMP1. Different forms of PfEMP1 are encoded by individual members of the large, multicopy var gene family. Only a single var gene is expressed at a time. Switching which var gene is expressed aids the parasite in its ability to evade clearance by the immune system and is referred to as antigenic variation. The mechanism by which the expression of individual var genes is regulated is poorly understood, however recent work has demonstrated that histone modifications play a significant role. Once a var gene is activated, it tends to remain active for many cellular divisions, a property referred to as "epigenetic memory." Studies have also shown that active transcription by RNA polymerase II is required for the maintenance of epigenetic memory of P. falciparum var genes, suggesting that the polymerase itself might play a role in maintaining the necessary chromatin modifications required for regulating var gene expression. In addition, it was recently shown that rodent parasites do not utilize epigenetic memory to control the expression of their virulence genes. Phylogenetic comparisons reveal an unusual expansion of the C-terminal domain (CTD) of the largest subunit of RNA pol II of primate parasites that is not found in parasites of rodents, birds or bats. Further, this region of RNA pol II has been shown to interact with chromatin modifying enzymes in higher eukaryotes, suggesting that it could be involved in maintenance of epigenetic memory. Interestingly, two specific histone modifying proteins, the histone methyltransferase PfSet2 and its cognate demethylase PfJmjC1, are similarly only found in primate parasites. Previous structural and biochemical work in yeast has shown that the phosphorylated form of the RNA pol II CTD can directly interact with Set2 to recruit it to actively transcribed genes, thus helping to maintain active chromatin at highly transcribed regions of the genome. Our hypothesis is that PfSet2 is recruited to transcriptionally active var genes through interactions with the phosphorylated CTD of P. falciparum RNA pol II to enforce epigenetic memory. The goal of this study is to determine if PfSet2 directly interacts with the CTD using biochemical and structural approaches, and to investigate the role of this interaction in the maintenance of epigenetic memory of var genes by introducing mutant forms of PfSet2 in P. falciparum. The long term goal is to determine the role of the interaction between the CTD and PfSet2 in promoting virulent mechanisms such as antigenic variation in P. falciparum. PUBLIC HEALTH RELEVANCE: Each year, millions of people worldwide are afflicted with P. falciparum malaria resulting in approximately a million deaths of mostly young children in sub-Saharan Africa. Previous work demonstrated that placement of an antigenic determinant called PfEMP1 on human red blood cells is essential for P. falciparum survival, and is regulated by a var gene switching mechanism that aids the parasite in its ability to evade the immune system. Understanding the regulation of this gene switching mechanism can lead to the development of more efficient drugs and possibly a vaccine against this acute and severe disease.