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 布氏锥虫会引起人类非洲锥虫病，这种疾病在不治疗的情况下往往是致命的。 治疗这种疾病的药物很少，大多数都有严重的副作用，而且很难 管理，而耐药布氏支原体感染一直在上升。此外，布氏毛滴虫还引起 非洲动物锥虫病，这是撒哈拉以南非洲的一个重大经济负担。它是 因此，进一步研究布氏毛滴虫的致病机理，为今后的发展寻找更好的靶点具有重要意义。 抗寄生虫剂。布氏毛滴虫经常交换其主要表面抗原Vsg，以逃避其哺乳动物宿主。 免疫反应。VSG转换是布鲁氏锥虫的主要致病机制。 建立并保持长期的感染。然而，VSG切换是如何在T. 布鲁塞仍不清楚。布氏毛滴虫有2500个VSG基因和假基因，它们都位于 亚端粒。然而，VSG仅通过亚端粒多顺反子VSG表达来表达 站点(ESS)。Vsg是任何ES中的最后一个基因，距离端粒重复序列不到2kb。布氏毛滴虫有多个 ESS具有非常相似的序列，但在任何时候只有一个ES是完全激活的，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 正在切换。大多数VSG基因的侧翼都有两个共同的序列，这为 DNA重组介导的VSG转换中VSG活性基因和沉默基因的重组 事件。首先，所有的VSG 3‘非编码区都有一个共同的14个核苷酸序列。还发现了长端粒序列 Es连锁的VSGs和VSGs的下游位于微染色体亚端粒。第二，大多数VSG的上游 基因是70bp的重复序列。Ess中的70bp重复序列可以是几个kb到几十个kb的长度。一直以来 研究表明，在70bp处引入DNA双链断裂会立即在活性DNA的上游重复 VSG基因使VSG切换率提高约250倍。此外，在70个碱基上发现了DNA断裂 在WT细胞中重复。因此，预计70BP重复完整性将显著影响VSG切换 频率然而，目前尚不清楚是否有任何蛋白质与这70个碱基重复序列特别相关， 尽管它们的序列是高度保守的，而且它们的相关蛋白质有望帮助 维护他们的诚信，并参与VSG切换的监管。在这个小项目中，我们的目标是 使用一种改进的“分离染色质片段的末端靶向蛋白质组学”(EPICh)方法分离 70个碱基重复染色质，并通过质谱学鉴定与70个碱基重复相关的蛋白质。我们会 验证70个碱基中确定的候选者，重复ePICh并启动他们的功能分析。我们的工作将 清楚地揭示是否有任何与70bp重复序列特定相关的蛋白质，这将使我们能够 研究70个碱基重复序列的完整性是如何维持的。我们的发现将开辟更好的新途径 了解布氏毛滴虫的致病机理，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.