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首页> 外文期刊>Epigenetics & Chromatin >Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes
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Silencing subtelomeric VSGs by Trypanosoma brucei RAP1 at the insect stage involves chromatin structure changes

机译：布鲁氏锥虫RAP1在昆虫阶段沉默亚端粒VSG涉及染色质结构变化

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BackgroundTrypanosoma brucei causes human African trypanosomiasisand undergoes antigenic variation to evade its mammalianhost immune attack. Throughout its life cycle,T. brucei is covered with glycoproteins on its cell surface.When infecting a mammalian host, bloodstream form(BF) T. brucei expresses bloodstream expression site(BES)-linked variant surface glycoproteins (VSGs) in amonoallelic fashion to ensure effectiveness of antigenicvariation. In the midgut of its insect vector, tsetse, procyclicform (PF) T. brucei expresses procyclins. After migrationto the tsetse’s salivary glands, T. brucei expressesmetacyclic VSGs (mVSGs) to prepare for mammalian hostinfection. Regulation of VSG expression/silencing is criticalfor T. brucei virulence and its development. Howeverthe underlying mechanism of VSG regulation is not fullyunderstood. Our lab identified a telomeric proteinTbRAP1 as a key regulator of BES-VSGs in BF [1]. In thisstudy we have further elucidated role of TbRAP1 in VSGregulation at PF stage and its possible underlying mechanismthrough chromatin remodeling.Materials and methodsWe depleted TbRAP1 in PF cells using an inducible RNAinterference (RNAi) approach. Quantitative RT-PCR wasperformed to analyze changes in steady state mRNA levelsof BES-lined and metacyclic VSGs after depletion ofTbRAP1. In order to test the effect of TbRAP1 depletionon chromatin structure, Formaldehyde-Assisted Isolationof Regulatory Elements (FAIRE) analysis and Micrococcalnucleasedigestion assays were performed.ResultsIn this study, we found that depletion of TbRAP1 by RNAiled to derepression of mVSGs at both the BF and PFstages. Similar to that observed in BF cells, silencing ofBES-linked VSGs in PF cells also depends on TbRAP1. Inaddition, we found that silencing of BES-linked VSGs byTbRAP1 is stronger in PF cells than that in BF cells.Furthermore, removal of TbRAP1 led to a loosened chromatinstructure at multiple loci in PF cells, particularly atthe BES loci, but not in BF cells, indicating that differentsilencing mechanisms are used at different stages and thatTbRAP1-mediated silencing may involve modulation ofchromatin structure in PF cells.ConclusionsOur observations confirm that TbRAP1 is a key VSG silencerand that TbRAP1 helps to determine the chromatinstructure in multiple loci throughout the genome, particularlyat BESs, at the insect stage.
机译：背景布鲁氏锥虫引起人类非洲锥虫病，并进行抗原变异，以逃避其哺乳动物宿主的免疫攻击。在整个生命周期中，T。布鲁氏菌在其细胞表面覆盖有糖蛋白。当感染哺乳动物宿主时，布鲁氏杆菌以单等位基因方式表达血流表达位点（BES）-连锁的变异表面糖蛋白（VSG），以确保抗原变异的有效性。在其昆虫媒介的中肠（采采蝇）中，前环形式（PF）的布鲁氏杆菌（T. brucei）表达环蛋白。迁移到采采蝇的唾液腺后，布鲁氏杆菌表达了metacyclic VSG（mVSG），为哺乳动物宿主感染做准备。 VSG表达/沉默的调节对于布鲁氏弧菌毒力及其发展至关重要。但是，对VSG监管的基本机制还没有完全了解。我们的实验室鉴定出端粒蛋白TbRAP1是高炉中BES-VSGs的关键调控因子[1]。在这项研究中，我们进一步阐明了TbRAP1在PF期VSG调节中的作用及其可能通过染色质重塑的潜在机制。材料和方法我们采用诱导RNA干扰（RNAi）方法消除了PF细胞中的TbRAP1。 TbRAP1耗尽后，进行定量RT-PCR分析BES内衬和间环VSGs稳态mRNA水平的变化。为了测试TbRAP1耗竭对染色质结构的影响，进行了甲醛辅助调节因子（FAIRE）分析和微球菌核酸酶消化试验。 。与BF细胞中观察到的相似，PF细胞中与BES相连的VSG的沉默也取决于TbRAP1。此外，我们发现在PF细胞中，TbRAP1对BES连锁VSG的沉默作用强于BF细胞。这表明在不同的阶段使用了不同的沉默机制，并且TbRAP1介导的沉默可能涉及PF细胞染色质结构的调节。 ，处于昆虫阶段。

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