BACKGROUND Imitation SWItch (ISWI) ATPase is the catalytic subunit in diverse chromatin remodeling complexes. These complexes modify histone-DNA interactions and therefore play a pivotal role in different DNA-dependent processes. In Trypanosoma cruzi, a protozoan that controls gene expression principally post-transcriptionally, the transcriptional regulation mechanisms mediated by chromatin remodeling are poorly understood. OBJECTIVE To characterise the ISWI remodeler in T. cruzi (TcISWI). METHODS A new version of pTcGW vectors was constructed to express green fluorescent protein (GFP)-tagged TcISWI. CRISPR-Cas9 system was used to obtain parasites with inactivated TcISWI gene and we determined TcISWI partners by cryomilling-affinity purification-mass spectrometry (MS) assay as an approximation to start to unravel the function of this protein. FINDINGS Our approach identified known ISWI partners [nucleoplasmin-like protein (NLP), regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP)], previously characterised in T. brucei, and new components in TcISWI complex [DRBD2, DHH1 and proteins containing a domain characteristic of structural maintenance of chromosomes (SMC) proteins]. Data are available via ProteomeXchange with identifier PXD017869. MAIN CONCLUSIONS In addition to its participation in transcriptional silencing, as it was reported in T. brucei, the data generated here provide a framework that suggests a role for TcISWI chromatin remodeler in different nuclear processes in T. cruzi, including mRNA nuclear export control and chromatin compaction. Further work is necessary to clarify the TcISWI functional diversity that arises from this protein interaction study.

中文翻译：

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表征克氏锥虫的ISWI染色质重塑剂。

背景技术模拟SWITCH（ISWI）ATPase是多种染色质重塑复合物中的催化亚基。这些复合物修饰组蛋白与DNA的相互作用，因此在不同的DNA依赖过程中起着关键作用。在克鲁斯锥虫（Trypanosoma cruzi）中，原生动物主要在转录后控制基因表达，人们对由染色质重塑介导的转录调控机制了解甚少。目的对T. cruzi（TcISWI）中的ISWI重塑器进行表征。方法构建了新版本的pTcGW载体，以表达带有绿色荧光蛋白（GFP）标签的TcISWI。CRISPR-Cas9系统用于获得带有灭活的TcISWI基因的寄生虫，我们通过冷冻铣削亲和纯化质谱法（MS）分析确定了TcISWI伴侣，以此来开始阐明该蛋白的功能。结果我们的方法确定了ISWI的已知合作伙伴[核纤溶酶样蛋白（NLP），染色体浓缩1样蛋白的调节剂（RCCP）和富含苯丙氨酸/酪氨酸的蛋白（FYRP）]，其以前以布鲁氏梭菌为特征，并且有新的成分。 TcISWI复合体[DRBD2，DHH1和包含染色体结构维持性结构域特征的蛋白质（SMC）蛋白质]。数据可通过ProteomeXchange获得，其标识符为PXD017869。主要结论除了布鲁氏杆菌中报道的参与转录沉默外，此处产生的数据提供了一个框架，暗示了TcISWI染色质重塑剂在克鲁氏酵母的不同核过程中的作用，包括mRNA核输出控制和染色质压实。