Trypanosoma cruzi cell cycle progression exhibits minimal variation in histone PTMs with unique histone H4 acetylation pattern

Abstract

Histones are crucial proteins in eukaryotic cells that undergo extensive posttranslational modifications (PTMs) such as methylation, acetylation, and phosphorylation, which are associated to chromatin structure, gene expression, DNA damage/repair and cell cycle. In Trypanosoma cruzi, the primary sequence of histones differs from that of other eukaryotes. Despite this, they display a vast range of PTMs, though their modulation throughout the cell cycle remains largely unexplored. In this study, we investigated the dynamic modulation of histone PTMs across G1/S, S, and G2/M phases of T. cruzi cell cycle using hydroxyurea- synchronized parasites. We applied a workflow that included histone derivatization, trypsin digestion followed by a high-resolution mass spectrometry and data independent analysis. Quantitative analysis of 141 histone peptide isoforms revealed that there are only minor variations in histone PTM levels throughout the cell cycle. The H3K76 trimethylation remained predominant throughout all phases, with an increase in monomethylation during G2/M. Additionally, hyperacetylation of the N-terminal region of histone H4 was observed, particularly at lysine residues 2, 5, and 10, suggesting their importance in cell cycle progression. Striking, acetylation of histone H4 at K2 and K5 increases during the S-phase, mirroring the H4K5acK12ac pattern observed in mammals, which are related to histone nuclear import and chromatin deposition.

Overall, the results suggest that the T. cruzi cell cycle maintains stable global levels of histone PTMs, relying on variations in only a few specific PTMs. Further investigations are warranted to elucidate the functional significance of these PTMs and their impact on cell cycle regulation and chromatin dynamics in T. cruzi.

Significance

Histone posttranslational modifications (PTMs) are key regulators of chromatin architecture and cellular processes such as gene expression and cell cycle control. In Trypanosoma cruzi, the etiological agent of Chagas disease, histones have a distinct primary structure compared to other eukaryotes, yet they display a wide variety of PTMs. This study provides a comprehensive analysis of histone PTM dynamics across the G1/S, S, and G2/M phases of the T. cruzi cell cycle, revealing that global histone PTM levels remain largely stable, with variations in a few specific marks. Notably, the study highlights the increased acetylation of histone H4 at lysines 2 and 5 during the S-phase, contrasting with the well-conserved acetylation at lysines 5 and 12 observed in mammals involved in nuclear import and chromatin assembly. These findings underscore the evolutionary divergence and functional specificity of histone modifications and provide a foundation for further investigations into their roles in parasite biology, with potential implications for understanding chromatin dynamics and identifying novel therapeutic targets.