The Histone Code of Toxoplasma gondii Comprises Conserved and Unique Posttranslational Modifications

Abstract

Epigenetic gene regulation has emerged as a major mechanism for gene regulation in all eukaryotes. Histones are small, basic proteins that constitute the major protein component of chromatin, and posttranslational modifications (PTM) of histones are essential for epigenetic gene regulation. The different combinations of histone PTM form the histone code for an organism, marking functional units of chromatin that recruit macromolecular complexes that govern chromatin structure and regulate gene expression. To characterize the repertoire of Toxoplasma gondii histone PTM, we enriched histones using standard acid extraction protocols and analyzed them with several complementary middle-down and bottom-up proteomic approaches with the high-resolution Orbitrap mass spectrometer using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and/or electron transfer dissociation (ETD) fragmentation. We identified 249 peptides with unique combinations of PTM that comprise the T. gondii histone code. T. gondii histones share a high degree of sequence conservation with human histones, and many modifications are conserved between these species. In addition, T. gondii histones have unique modifications not previously identified in other species. Finally, T. gondii histones are modified by succinylation, propionylation, and formylation, recently described histone PTM that have not previously been identified in parasitic protozoa. The characterization of the T. gondii histone code will facilitate in-depth analysis of how epigenetic regulation affects gene expression in pathogenic apicomplexan parasites and identify a new model system for elucidating the biological functions of novel histone PTM.

O -fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

“…AAL binding is also found in proximity to euchromatin (Fig. S7C), as defined by antibodies against two histone modifications associated with active transcription, H3K9Ac and H3K4Me3 (9). Last, two predicted FG-NupsNup68, which is enriched by AAL, and Nup67, which is not-were expressed with a C-terminal YFP under their endogenous promoters ( Fig.…”

“…Transcriptome analyses have revealed that a large percentage of mRNAs show life stage-specific expression (4) and/or cell cycle regulation (5). Recent studies have increased our understanding of gene expression in T. gondii by identifying the AP2 family of transcription factors (6)(7)(8) and by describing posttranslational modifications (PTMs) of histones and some of the enzymes responsible for them (9)(10)(11). However, little is known about protein organization at the nuclear periphery, a subnuclear compartment that plays a critical role in transcriptional regulation in many eukaryotes.…”

Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii

“…H2A.Z and H2B.Z have been shown to be hyper-acetylated in the N-terminal [30]. Acetylation is one of the most prominent modifications influencing gene expression [31, 32] as neutralization of the positively charged histone N-terminal tails leads to chromatin decondensation, thereby enhancing transcriptional activity [32].…”

“…Acetylation is one of the most prominent modifications influencing gene expression [31, 32] as neutralization of the positively charged histone N-terminal tails leads to chromatin decondensation, thereby enhancing transcriptional activity [32]. In other organisms, as well as T. gondii , H2A.Z with its hyperacetylated N-terminal region, is preferentially present in regions of the genome correlating with transcriptional activation [33], and this should also be true for H2B.Z, where the N-terminal tail is acetylated on lysines 3, 8, 13, 14, and 18 [30]. Similar results were also observed in P. falciparum [34, 35].…”

Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3

“…Analysis of the post-translational modifications of histones was conducted by combining two protocols (Nardelli et al, 2013;Shechter et al, 2007). Embryos of the specified developmental stage ( preDII, DII, postDII; 18-36 dpf ) were collected and rinsed in 0.03% hypochlorite for 5 min to remove external chorionic debris and microorganisms, and then rinsed once with diH 2 O.…”