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端结构域和组蛋白甲基转移酶之间的相互作用在介导疟疾毒力基因表观遗传记忆中的作用恶性疟原虫是导致最急性和严重形式的人类疟疾的原因。这种原生动物寄生虫感染它的人类宿主的循环红细胞，在被感染的细胞表面放置主要的毒力和抗原决定因素，一种叫做PfEMP1的粘附蛋白。不同形式的PfEMP1是由多拷贝var基因家族的个体成员编码的。一次只表达一个var基因。表达var基因的转换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。个体var基因表达调控的机制尚不清楚，但最近的研究表明，组蛋白修饰起着重要作用。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的活性转录是维持恶性疟原虫var基因表观遗传记忆所必需的，这表明该聚合酶本身可能在维持调节var基因表达所需的必要染色质修饰中发挥作用。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末端结构域（CTD）的异常扩展，这在啮齿动物、鸟类或蝙蝠的寄生虫中没有发现。此外，该区域的RNA pol II已被证明与高级真核生物的染色质修饰酶相互作用，表明它可能参与维持表观遗传记忆。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，类似地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，将其招募到转录活跃的基因中，从而帮助维持基因组高转录区域的活性染色质。我们的假设是，通过与恶性疟原虫RNA pol II的磷酸化CTD相互作用，PfSet2被募集到具有转录活性的var基因上，以加强表观遗传记忆。本研究的目的是通过生化和结构方法确定PfSet2是否直接与CTD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的毒力机制（如抗原变异）中的作用。

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.