Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi

Abstract

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi

“…Protocol 1 was based on (20) whereas protocol 2 was based on (21). Both are described in details at (22). Protocol 3 was described in (23), with a few modifications.…”

“…In-gel tryptic protein digestion was performed as described in (24). Insolution digestion was described in details at (22). Briefly, after TCA precipitation, 150 g of protein extracts were reduced with 5 mM of DTT for 30 min at room temperature, alkylated with 14 mM of iodoacetamide in the dark for 30 min and digested with 0.5 g of Lys-C (Promega, Madison, Wisconsin) for 4 h at 37°C, under agitation (900 rpm).…”

Quantitative chromatin proteomics reveals a dynamic histone post-translational modification landscape that defines asexual and sexual Plasmodium falciparum parasites

“…Histone acetylation is associated with gene activation in a transcriptionally permissive state, whereas methylation results in repression or activation, in a position-dependent manner 2 . Although histones and their modifying proteins are conserved between different organisms, they contain alternative sites for modification within early-branched Eukarya, mediated by divergence of the modifying proteins 3, 4 . This reveals evolutionary unique functions for these histone PTMs in gene expression regulation and other chromatin-mediated processes involved during development 5–9 .…”

Specialising the parasite nucleus: Pores, lamins, chromatin, and diversity

“…cruzi trypomastigotes attach and actively invade mammalian cells but do not divide, these cells mainly restrict synthetic activity to maintaining surface components that interface with the host cell [101]. These changes are probably consequences of a low state of transcription [99] and the presence of unique post-transcriptional modification of histones and proteins [102]. When T .…”