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)：RNAPolII的C-末端区域和组蛋白甲基转移酶之间的相互作用在调节疟疾毒力基因表观遗传记忆中的作用恶性疟原虫是最急性和最严重的人类疟疾的罪魁祸首。这种原生动物寄生虫感染其宿主的循环红细胞，在受感染的细胞表面放置主要毒力和抗原决定簇，一种名为PfEMP1的粘连蛋白。不同形式的PfEMP1由大的、多拷贝的var基因家族的单个成员编码。一次只有一个var基因表达。切换表达哪个var基因有助于寄生虫逃避免疫系统的清除，被称为抗原变异。目前对单个var基因表达调控的机制知之甚少，但最近的研究表明组蛋白修饰起着重要的作用。一旦var基因被激活，它往往会在许多细胞分裂中保持活跃，这一特性被称为“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，啮齿动物寄生虫并不利用表观遗传记忆来控制其毒力基因的表达。系统发育比较显示，灵长类寄生虫RNA polII最大亚基的C末端结构域(CTD)异常扩大，这在啮齿动物、鸟类或蝙蝠的寄生虫中没有发现。此外，RNA PolII的这个区域已经被证明与高等真核生物中的染色质修饰酶相互作用，这表明它可能参与了表观遗传记忆的维持。有趣的是，两种特殊的组蛋白修饰蛋白，组蛋白甲基转移酶PfSet2和它的同源去甲基酶PfJmjC1，同样只在灵长类寄生虫中发现。此前在酵母中的结构和生化研究表明，磷酸化形式的RNA polII CTD可以直接与Set2相互作用，将其招募到活跃转录的基因中，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，PfSet2通过与恶性疟原虫RNA PolII的磷酸化CTD相互作用而被招募到转录活性的var基因，以加强表观遗传记忆。本研究的目的是确定PfSet2是否通过生化和结构方法直接与CTD相互作用，并通过在恶性疟原虫中引入突变形式的PfSet2来研究这种相互作用在维持var基因的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毒力机制(如抗原变异)中的作用。 公共卫生意义：每年，全世界有数百万人感染恶性疟原虫，导致撒哈拉以南非洲约100万人死亡，其中大部分是幼儿。以前的工作表明，PfEMP1抗原决定簇在人红细胞上的放置对恶性疟原虫的生存是必不可少的，并受一种var基因切换机制的调节，该机制帮助疟原虫逃避免疫系统。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，并可能开发出预防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.