Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite.

Significance

Trypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.

中文翻译：

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克氏锥虫组蛋白翻译后修饰定量分析的改进：通过寄生虫的元细胞发生和生命周期研究表观遗传标记的变化。

锥虫组蛋白 N 端序列与其他真核生物非常不同，尽管它们仍然被翻译后修饰 (PTM) 修饰。在这里，我们使用高度稳健的工作流程，使用基于质谱（基于 MS）的数据独立采集 (DIA)分析寄生虫克氏锥虫中的组蛋白 PTM。我们通过修改软件 EpiProfile 2.0 调整了分析寄生虫组蛋白序列的工作流程，提高了肽和 PTM 量化的准确性。该工作流程现在可应用于 141 种 克氏锥虫修饰的组蛋白肽的研究，我们使用这些肽研究了组蛋白 PTM 沿元细胞发生和T. cruzi生命周期的动态. 组蛋白乙酰化和甲基化的全球水平随着元细胞发生而波动，但是在寄生虫生命形式之间观察到最关键的差异。检测到超过 66 个组蛋白 PTM 变化。引人注目的是，后循环锥体鞭毛体的组蛋白 PTM 模式与表皮鞭毛体更相似，而不是细胞锥体鞭毛体。最后，我们强调了 H4 N 末端和 H3K76 的变化，讨论了它们对锥虫生物学的影响。总而言之，我们优化了一个工作流程，该工作流程很容易适用于克氏锥虫中组蛋白 PTM 的分析，并生成了一个数据集，可以阐明染色质修饰在该寄生虫中的作用。

意义

锥虫是单细胞寄生虫，具有不同的组蛋白序列、无染色体浓缩和特殊的基因组/基因调控。基因从不同的多顺反子区域转录，转录后基因调控在转录本和蛋白质水平的建立中起主要作用。在这方面，他们的组蛋白装饰有多个 PTM 这一事实引发了有关其作用的有趣问题。此外，这种双基因生物必须适应不同的环境，相应地改变其新陈代谢。由于新陈代谢和表观遗传学密切相关，因此对锥虫中组蛋白 PTM 的研究可能会启发这种引人注目但尚未完全理解的相互作用。从生物医学的角度来看，