Identify 70 bp repeat-associated chromatin components by End-targeting Proteomics of Isolated Chromatin segments (PICh) and initiate their functional characterization

项目概要 布氏锥虫会引起人类非洲锥虫病，如果不治疗，这种病通常会致命。 治疗这种疾病的药物很少，大多数副作用严重且难以治疗。 布氏锥虫耐药性感染呈上升趋势。此外，T. brucei 还会导致 非洲动物锥虫病，已成为撒哈拉以南非洲地区的重大经济负担。它是 因此，进一步研究布氏锥虫的发病机制并确定未来发展的更好目标非常重要 抗寄生虫剂。布氏锥虫定期改变其主要表面抗原 VSG，以逃避哺乳动物宿主 免疫反应。 VSG 转换是一种主要的发病机制，使布氏锥虫能够 建立并维持长期感染。然而，VSG 切换是如何在 T 中自然启动的。 布鲁斯还不清楚。 T. brucei 有超过 2,500 个 VSG 基因和假基因，全部位于 亚端粒。然而，VSG 仅由亚端粒多顺反子 VSG 表达表达 站点（ES）。 VSG 是任何 ES 中的最后一个基因，距离端粒重复序列在 2 kb 范围内。 T. brucei 有多种 ES 的序列非常相似，但任何时候只有一个 ES 完全活跃，导致 VSG 单一类型 表达于细胞表面。 DNA 重组已被证明是 VSG 的主要途径 切换。大多数 VSG 基因两侧有两个共同序列，这提供了序列同源性 DNA重组介导的VSG转换中活性和沉默VSG基因之间的重组 事件。首先，所有 VSG 3’UTR 都有一个共同的 14 nt 序列。还发现了长端粒序列 ES 连接的 VSG 和微型染色体亚端粒处的 VSG 下游。二、大多数VSG的上游 基因有 70 bp 重复。 ES 中的 70 bp 重复序列可以有几 kb 到几十 kb 长。已经是 结果表明，在紧邻活性物质上游的 70 bp 重复序列中引入 DNA 双链断裂 VSG 基因将 VSG 转换率提高约 250 倍。此外，在 70 bp 处发现 DNA 断裂。 在 WT 细胞中重复。因此，70 bp 重复完整性预计会显着影响 VSG 切换 频率。然而，尚不清楚是否有任何蛋白质与 70 bp 重复序列特异性相关， 尽管它们的序列高度保守，并且它们的相关蛋白预计会有所帮助 保持其完整性并参与 VSG 切换的监管。在这个小项目中，我们的目标是 使用改进的“分离染色质片段的末端靶向蛋白质组学”（ePICh）方法来分离 70 bp 重复染色质并通过质谱鉴定与 70 bp 重复相关的蛋白质。我们会 验证 70 bp 重复 ePICh 中识别的候选者并启动其功能分析。我们的工作将 清楚地揭示是否有任何蛋白质与 70 bp 重复序列特异性相关，这将使我们能够 研究如何保持 70 bp 重复序列的完整性。我们的研究结果将为更好地开展工作开辟新途径 了解布氏锥虫的发病机制并有助于最终根除这种寄生虫。

Project Summary Trypanosoma brucei causes human African trypanosomiasis, which is frequently fatal without treatment. Few drugs are available for treating this disease, most of which have severe side-effects and are difficult to administer, while drug-resistant T. brucei infection has been on the rise. In addition, T. brucei also causes animal African trypanosomiasis, which has been a significant economic burden in sub-Sahara Africa. It is therefore important to further study T. brucei pathogenesis and identify better targets for future development of anti-parasite agents. T. brucei regularly switches its major surface antigen, VSG, to evade its mammalian host immune response. VSG switching is a major pathogenesis mechanism that enables T. brucei to establish and maintain a long-term infection. However, how VSG switching is initiated naturally in T. brucei is still not clear. T. brucei has >2,500 VSG genes and pseudogenes, all of which are located at subtelomeres. However, VSGs are expressed exclusively from subtelomeric polycistronic VSG expression sites (ESs). VSG is the last gene in any ES and within 2 kb from the telomere repeats. T. brucei has multiple ESs with very similar sequences, but only one ES is fully active at any time, resulting in a single type of VSG being expressed on the cells surface. DNA recombination has been shown to be a major pathway for VSG switching. Most VSG genes are flanked by two common sequences, which provide sequence homology for recombination between the active and a silent VSG gene in DNA recombination-mediated VSG switching events. First, all VSG 3’UTR has a common 14 nt sequences. Long telomere sequences are also found downstream of ES-linked VSGs and VSGs at minichromosome subtelomeres. Second, upstream of most VSG genes are 70 bp repeats. The 70 bp repeats in ESs can be several kb to several tens kb long. It has been shown that introducing a DNA double strand break in the 70 bp repeats immediately upstream of the active VSG gene increases the VSG switching rate for ~ 250 fold. In addition, DNA breaks are found in the 70 bp repeats in WT cells. Therefore, 70 bp repeat integrity is expected to significantly affect VSG switching frequency. However, it is unknown whether any proteins specifically associate with the 70 bp repeats, even though their sequences are highly conserved, and their associated proteins are expected to help maintain their integrity and to participate in the regulation of VSG switching. In this small project, we aim to use an improved “end-targeting proteomics of isolated chromatin segments” (ePICh) approach to isolate the 70 bp repeat chromatin and identify proteins associated with the 70 bp repeats by mass spectrometry. We will validate candidates identified in the 70 bp repeat ePICh and initiate their functional analysis. Our work will reveal clearly whether any proteins specifically associate with the 70 bp repeats, which will allow us to investigate how 70 bp repeats integrity is maintained. Our findings will open up new avenues for better understanding of the T. brucei pathogenesis and contribute to eradicate this parasite eventually.