Exploring the virulence gene interactome withCRISPR/dCas9 in the human malaria parasite
Exploring the virulence gene interactome withCRISPR/dCas9 in the human malaria parasite
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DOI：
10.15252/msb.20209569
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发表时间：
2020-08-01
影响因子：
9.9
通讯作者：
Scherf, Artur
中科院分区：
文献类型：
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作者：
Bryant, Jessica M.;Baumgarten, Sebastian;Scherf, Artur
var多基因家族的相互排斥表达是恶性疟原虫免疫逃避和致病的关键，但很少有因素被证明发挥直接作用。我们采用了基于CRISPR的蛋白质组学方法来识别与天然染色质背景中的可变基因相关的新因子。将催化失活的Cas9（“dCas 9”）靶向于不同基因调控元件，免疫沉淀，并用质谱分析。已知和新的因素，包括结构蛋白，DNA解旋酶，染色质重塑丰富。PfISWI是一种在thevargene启动子处富集的进化上不同的假定染色质重塑物，其功能表征揭示了其在转录激活中的作用。PfISWI的蛋白质组学鉴定了几种在var基因启动子富集的蛋白质，如乙酰辅酶A合成酶，一种假定的MORC蛋白和ApiAP 2转录因子。这些发现验证了CRISPR/dCas 9蛋白质组学方法，并定义了一种新的vargene相关染色质复合物。这项研究建立了一个工具，有针对性的染色质纯化未改变的基因组位点，并确定新的染色质相关因子可能参与转录控制和/或染色质组织的毒力基因在人类疟疾寄生虫。
Mutually exclusive expression of thevarmultigene family is key to immune evasion and pathogenesis inPlasmodium falciparum, but few factors have been shown to play a direct role. We adapted aCRISPR-based proteomics approach to identify novel factors associated withvargenes in their natural chromatin context. Catalytically inactive Cas9 ("dCas9") was targeted tovargene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins,DNAhelicases, and chromatin remodelers. Functional characterization ofPfISWI, an evolutionarily divergent putative chromatin remodeler enriched at thevargene promoter, revealed a role in transcriptional activation. Proteomics ofPfISWIidentified several proteins enriched at thevargene promoter such as acetyl-CoA synthetase, a putativeMORCprotein, and an ApiAP2 transcription factor. These findings validate theCRISPR/dCas9 proteomics method and define a newvargene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.