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

通过分离染色质片段 (PICh) 的末端靶向蛋白质组学鉴定 70 bp 重复相关染色质成分，并启动其功能表征

• 批准号: 10417263
• 负责人: Bibo Li
• 金额: $ 7.43万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-04 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10417263
• 关键词: 3' Untranslated Regions; Affect; Africa; African; African Trypanosomiasis; Animals; Binding; Binding Proteins; Cell surface; Chromatin; Complex; Conserved Sequence; Country; Coupled; DNA; DNA Double Strand Break; Development; Disease; Drug resistance; Economic Burden; Event; Fluorescent in Situ Hybridization; Frequencies; Future; Gene Conversion; Generations; Genes; Genetic Recombination; Human; Immune response; Immunofluorescence Immunologic; Immunologic Surveillance; In Situ; Infection; Link; Mass Spectrum Analysis; Mediating; Messenger RNA; Parasites; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Play; Proteins; Proteomics; Protocols documentation; Pseudogenes; RNA Interference; RNA Probes; Regulation; Role; Sahara; Sequence Homology; Site; Surface Antigens; Time; Trypanosoma brucei brucei; Work; cell growth; cell type; derepression; genetic information; improved; mortality; side effect; telomere

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.

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，大多数都有严重的副作用并且很难治愈。 布氏锥虫感染也呈上升趋势。 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。 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，以逃避其哺乳动物宿主。 VSG 转换是 T. brucei 的主要发病机制。 然而，VSG 转换是如何在 T 中自然启动的。 brucei 仍不清楚 T. brucei 有超过 2,500 个 VSG 基因和假基因，所有这些基因都位于 然而，VSG 仅由亚端粒多顺反子 VSG 表达表达。 VSG 是所有 ES 中的最后一个基因，距离 T. brucei 重复序列 2 kb 以内。 ES 具有非常相似的序列，但任何时候只有一个 ES 完全活跃，从而导致单一类型的 VSG DNA 重组已被证明是 VSG 的主要途径。 大多数 VSG 基因两侧都有两个共同序列，这为转换提供了序列同源性。 DNA重组介导的VSG转换中活性和沉默VSG基因之间的重组 首先，所有 VSG 3'UTR 都有一个共同的 14 nt 序列。 其次，大多数 VSG 的上游。 ES 中的 70 bp 重复序列可以有几 kb 到几十 kb 长。 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。 因此，70 bp 重复完整性预计会显着影响 VSG 转换。 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 在这个小项目中，我们的目标是保持其完整性并参与 VSG 切换的监管。 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。 验证 70 bp 重复 ePICh 中确定的候选者并启动其功能分析。 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实现这一目标开辟新途径。 了解布氏锥虫的发病机制并有助于最终根除这种寄生虫。