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

项目摘要 布氏锥虫引起人类非洲锥虫病，如果不治疗，往往是致命的。 治疗该病的药物很少，大多数药物副作用严重，难以治愈。 而耐药T.布氏杆菌感染呈上升趋势。此外，T.布氏杆菌还引起 动物非洲锥虫，这一直是撒哈拉以南非洲的重大经济负担。是 因此，进一步研究T.布氏病发病机制，并确定更好的目标，为今后的发展， 抗寄生虫剂。T.布氏杆菌有规律地转换其主要表面抗原VSG，以逃避哺乳动物宿主 免疫反应VSG转换是T.布鲁塞岛 建立并维持长期感染。然而，如何VSG开关自然启动T。 布鲁塞还不清楚。T.布氏杆菌有超过2,500个VSG基因和假基因，所有这些基因都位于 亚端粒然而，VSG仅从亚端粒多顺反子VSG表达中表达， 站点（ES）。VSG是任何ES中的最后一个基因，距离端粒重复序列2kb以内。T.布氏杆菌有多种 ES具有非常相似的序列，但在任何时候只有一个ES完全激活，从而导致单一类型的VSG 在细胞表面表达。DNA重组已被证明是VSG的主要途径 切换大多数VSG基因的侧翼是两个共同的序列，这为VSG基因提供了序列同源性。 在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具有共同的14 nt序列。长端粒序列也被发现 ES连锁的VSG下游和微小染色体亚端粒处的VSG。其次，大多数VSG的上游 基因是70 bp重复序列。ES中的70 bp重复序列可以是几kb到几十kb长。已经 显示在活性蛋白的上游直接引入70 bp重复序列中的DNA双链断裂， VSG基因使VSG转换率增加约250倍。此外，在70 bp的DNA片段中发现了DNA断裂， 在WT细胞中重复。因此，预期70 bp重复完整性显著影响VSG转换 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， 尽管它们的序列是高度保守的，它们的相关蛋白质有望帮助 保持其完整性，并参与VSG切换的调节。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。我们将 验证在70 bp重复ePICh中鉴定的候选物，并启动其功能分析。我们的工作将 清楚地揭示了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。我们的发现将为更好地 对T.布氏杆菌致病性，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.