Characterize functions of T. brucei RAP1 and TRF in antigenic variation and telom

描述(申请人提供)：布氏锥虫是一种原生动物寄生虫，可引起非洲人锥虫病和牛的纳加纳。在哺乳动物宿主中，布鲁氏血吸虫经历抗原变异，并有规律地切换其表面抗原，变异表面糖蛋白(VSG)，以逃避宿主的免疫攻击。尽管布氏锥虫有一千多个VSG基因和假基因，但VSG是以严格的单等位基因从亚端粒座位上唯一表达的，这确保了VSG切换的有效性，并使其效率最大化。因此，VSG的转换和单等位基因VSG的表达在布鲁氏毛滴虫的致病过程中是必不可少的。端粒与VSG的表达部位相邻，长期以来被认为在VSG的表达调控中起重要作用。事实上，已有研究表明，在酵母、人类和布氏支原体细胞中，端粒形成一种异染色质结构，影响针对亚端粒的报告基因的转录。特别是在酵母中，这种端粒沉默已被证明依赖于端粒蛋白RAP1。为了探索端粒在VSG表达和开关调节中的功能，我们一直致力于鉴定布氏毛滴虫端粒特异蛋白并对其功能进行鉴定。我们克隆了第一个布鲁氏毛滴虫端粒结合蛋白tbTRF。更重要的是，我们最近在以tbTRF为诱饵的酵母双杂交筛选中发现了布鲁氏锥虫RAP1，证实了tbRAP1是端粒复合体的内在成分，并证明了它对于沉默布鲁氏锥虫细胞中的亚端粒VSGs是必不可少的。这一发现揭示了布氏毛滴虫端粒在表面抗原表达控制中的关键作用，并确定tbRAP1是抗寄生虫药物的潜在靶点。我们的发现还表明，布氏毛滴虫与包括恶性疟原虫和光滑毛滴虫在内的其他几种病原体相似，其中端粒沉默在毒力基因表达的调节中发挥着重要作用。我们计划进一步研究端粒蛋白，特别是tbRAP1和tbTRF如何调节VSG的表达和开关。这项研究将有助于最终根除布氏毛滴虫和其他类似的微生物病原体。我们提出了几种方法来进一步研究tbRAP1和tbTRF的功能。首先，我们的观察表明，tbRAP1的端粒定位对其VSG沉默功能至关重要。因此，我们假设，如果tbRAP1直接与端粒DNA结合，这一活动将是VSG沉默的关键。然而，如果tbRAP1缺乏DNA结合活性，tbRAP1和tbTRF之间的相互作用以及tbTRF的端粒结合功能将是将tbRAP1锚定到端粒上所必需的。我们将使用体外方法在特定目标1中测试这些假设。这些研究将揭示tbRAP1介导的沉默的关键机制，并阐明tbRAP1和其他RAP1同源物的DNA结合和蛋白质相互作用功能的相似和独特的特征。因此，我们的工作将有助于确定抗寄生虫药物的潜在靶点。在具体目标2中，我们旨在进行一系列体内遗传分析，以进一步了解tbRAP1的S功能。首先，我们假设tbRAP1不仅参与VSG沉默控制，而且还影响VSG切换率，这将在AIM 2.1中进行测试。其次，我们将阐明tbRAP1不同功能之间的关系，并使用系统遗传学方法在AIM 2.2中确定tbRAP1的哪些结构域对于VSG的表达和/或开关调控是必不可少的。这些研究将有助于揭示tbRAP1的S在抗原变异中的作用机制。第三，我们假设tbRAP1通过调节染色质结构而发挥其沉默作用。因此，我们将检查tbRAP1是否优先与沉默的染色质相关，以及tbRAP1的缺失是否导致去抑制的ES的染色质结构发生任何变化。最后，我们假设tbRAP1与其他未知的辅助因子共同作用，建立/维持亚端粒基因座的沉默结构。为了寻找在抗原变异中也起重要作用的tbRAP1相互作用因子，我们旨在通过序贯免疫沉淀法鉴定tbRAP1蛋白复合体的组成。研究其他因素在同一tbRAP1介导的沉默途径中的作用将有助于我们更好地理解其潜在的机制。在与tbRAP1相互作用的候选基因中，我们可能发现参与VSG沉默/转换的下游效应因子，以及参与调节tbRAP1表达、稳定性或活性的蛋白质。识别与抗原变异有关的新因素也为抗寄生虫药物提供了更多潜在的靶点，并有助于最终消除这种寄生虫。

DESCRIPTION (provided by applicant): Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis in humans and nagana in cattle. In mammalian hosts, bloodstream form (BF) T. brucei undergoes antigenic variation and regularly switches its surface antigen, variant surface glycoprotein (VSG), to evade the host's immune attack. Although T. brucei has more than a thousand VSG genes and pseudogenes, VSG is expressed exclusively from subtelomeric loci in a strictly monoallelic manner, which ensures effectiveness of VSG switching and maximizes its efficiency. Therefore, VSG switching and monoallelic VSG expression are essential for T. brucei pathogenesis. Telomeres, being adjacent to the expression sites of VSGs, have long been proposed to play an important role in VSG expression regulation. Indeed, it has been shown that in yeast, human, and T. brucei cells, telomeres form a heterochromatic structure that affects the transcription of reporter genes targeted to subtelomeres. Particularly in yeast, this telomeric silencing has been shown to depend on telomere protein RAP1. To explore telomere functions in VSG expression and switching regulation, we have been focusing on identification of T. brucei telomere-specific proteins and characterization of their functions. We have cloned the first T. brucei telomere-binding protein, tbTRF. More importantly, we have recently identified T. brucei RAP1 in a yeast 2-hybrid screen using tbTRF as bait, confirmed that tbRAP1 is an intrinsic component of the telomere complex, and demonstrated that it is essential for silencing subtelomeric VSGs in BF T. brucei cells. This discovery reveals T. brucei telomere as a key player in surface antigen expression control and identifies tbRAP1 as a potential target for anti-parasite drugs. Our finding also shows that T. brucei is similar to a couple of other pathogens including P. falciparum and C. glabrata, in which telomeric silencing plays an important role in regulation of virulence gene expression. We plan to further study how telomere proteins, particular tbRAP1 and tbTRF, regulate VSG expression and switching. This study would be helpful for eventual eradication of T. brucei and other similar microbial pathogens. We propose several approaches to further study the functions of tbRAP1 and tbTRF. First, our observations suggest that localization of tbRAP1 to the telomere is crucial for its VSG silencing function. We therefore hypothesize that if tbRAP1 binds telomere DNA directly, this activity would be critical for VSG silencing. However, if tbRAP1 lacks any DNA binding activity, the interaction between tbRAP1 and tbTRF and the telomere binding function of tbTRF would be essential for anchoring tbRAP1 to the telomere. We will test these hypotheses in Specific Aim 1 using in vitro approaches. These studies will reveal a key mechanism for tbRAP1-mediated silencing and elucidate similar and unique features of the DNA binding and protein-protein interaction functions of tbRAP1 and other RAP1 homologs. Our work would therefore help to identify potential targets for anti-parasite drugs. In Specific Aim 2, we aim to carry out a series of in vivo genetic analyses to further understand tbRAP1's function. First, we hypothesize that tbRAP1 not only participates in VSG-silencing control but also influences VSG switching rates, which will be tested in Aim 2.1. Second, we will elucidate the relationship between different functions of tbRAP1 and to determine which domains of tbRAP1 are essential for VSG expression and/or switching regulation using systematic genetic approaches in Aim 2.2. These studies will help to reveal the underlying mechanisms of tbRAP1's function in antigenic variation. Third, we hypothesize that tbRAP1 applies its silencing effect by modulating the chromatin structure. We will therefore examine whether tbRAP1 preferentially associates with the silent chromatin and whether loss of tbRAP1 causes any changes in the chromatin structure of the derepressed ESs. Last, we hypothesize that tbRAP1 works with other unknown co-factors to establish/maintain the silencing structure at subtelomeric loci. In order to search for tbRAP1-interacting factors that also play important roles in antigenic variation, we aim to identify components of tbRAP1 protein complex by sequential immunoprecipitation. Studying functions of other factors in the same tbRAP1-mediated silencing pathway will help us to better understand the underlying mechanisms. Among the tbRAP1-interacting candidates, we may identify downstream effectors involved in VSG silencing/switching and proteins involved in the regulation of the expression, stability, or activity of tbRAP1. Identification of novel factors involved in antigenic variation also provides more potential targets for anti-parasite drugs and helps for eventual elimination of this parasite.