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.

Importance: Toxoplasma gondii is among the most common parasitic infections in humans. The transition between the different stages of the T. gondii life cycle are essential for parasite virulence and survival. These differentiation events are accompanied by significant changes in gene expression, and the control mechanisms for these transitions have not been elucidated. Important mechanisms that are involved in the control of gene expression are the epigenetic modifications that have been identified in several eukaryotes. T. gondii has a full complement of histone-modifying enzymes, histones, and variants. In this paper, we identify over a hundred PTM and a full repertoire of PTM combinations for T. gondii histones, providing the first large-scale characterization of the T. gondii histone code and an essential initial step for understanding how epigenetic modifications affect gene expression and other processes in this organism.

FIG 1

Identification of T. gondii histone posttranslational modifications. (A) Coomassie blue-stained gel showing a representative histone acid extraction sample used for PTM analysis. Relative molecular standards and positions of histones are marked. (B) Scheme of the combined techniques used to identify PTM in T. gondii. Tachyzoites were grown, collected, and purified from human fibroblasts as described in Materials and Methods and Text S1 in the supplemental material. Parasites were lysed, and intact nuclei were purified. The histones were extracted from chromatin under acidic conditions. MS data collection was performed on an LTQ Orbitrap Velos system, and spectra were searched in a custom, combined human Toxoplasma database (60) using Mascot.

FIG 2

Summary of PTM identified on Toxoplasma gondii canonical histones. PTM on canonical histones are shown in comparison with P. falciparum (Pf) and Homo sapiens (Hs) histones. Squares in different colors represent the modifications on three organisms. PTM from Plasmodium were obtained from the literature (–16). PTM from human cells were obtained from available databases (http://www.uniprot.org, http://www.cellsignal.com, and http://www.actrec.gov.in/histome [61]). Recently identified modifications, such as succinylation, O-GlcNAcylation, propionylation, formylation, and crotonylation, were obtained from the literature (–4, 47, 48). The gray cylinders indicate the globular domain. Numbers above the squares represent the numbers of methyl groups (“1,”“2,” and “3” indicate mono-, di-, and trimethylation, respectively), while those below the sequence represent the amino acid position in human histones. See Fig. S1 for aligned sequences and the supplemental material for combinations of PTM identified on peptides. In some cases, the exact residue modified by a PTM was not identified. These peptides are not indicated in this schematic but are listed in the supplemental material, which lists each of the peptides identified by mass spectrometry.

FIG 3

Overview of PTM identified on T. gondii histone variants. PTM on canonical and histone variants are shown in comparison with P. falciparum. Identical amino acids are represented in gray. Squares in different colors represent the modifications on Toxoplasma and Plasmodium. PTM from Plasmodium were obtained from available published literature (–16). The gray cylinders indicate the approximate locations of the globular domain. Numbers below the Toxoplasma sequence represent the amino acid position in this parasite, while “1,” “2,” and “3” above indicate mono-, di-, and trimethylation, respectively. See Fig. S2 for a complete alignment of variant histones and elsewhere in the supplemental material for combinations of PTM identified on peptides. In some cases, the exact residue modified by a PTM was not identified. These peptides are not indicated in this schematic but are listed in the supplemental material, which lists each of the peptides identified by mass spectrometry.

FIG 4

Representative MS spectra for T. gondii histone H3 peptide from positions 1 to 49. (A) Spectra showing the abundance of 0 to 12 methyl groups present on this peptide (charge: +9). The majority of the identified peptides show 5 methyl groups on lysines and/or arginines. (B) An example of a methylated TgH3 peptide analyzed by MS/MS shows methylation on K4, trimethylation on K36, and methylation on either R17 or K18. Residues that distinguish TgH3 from HsH3 are S22, M31, S32, and I35 (see also the supplemental material).

FIG 5

Mass spectrometry analysis of PTM for a T. gondii-specific H3 peptide. T. gondii peptide from amino acids 9 to 36 (KSTGGKAPRKQLASKAARKSAPMSGGIK) is unique to T. gondii histone 3. The serine at position 22 in T. gondii histone 3 is a threonine in human H3 (see Fig. S1). Seven PTM were identified in this TgH3 sequence (K9ac, K14ac, R17me, K18ac, K23ac, R26me2, and K27ac). The MS/MS spectra obtained with HCD of the +3 m/z ion at 1021.8967 is shown.

FIG 6

T. gondii histone H3 is succinylated and ubiquitinated. (A) MS/MS spectrum of the succinylated H3 from T. gondii peptide from positions 117 to 128 (VTIMPKDIQLAR). A succinylated lysine at position 122 was identified. The succinyl mark has a mass of 100.016 Da, as represented in the figure. The MS/MS spectra were obtained with HCD of the +2 m/z ion at 742.9096. (B) MS/MS spectrum of the ubiquitinated H3 from T. gondii peptide from positions 84 to 116 (FQSSAVLALQEAAEAYLVGLFEDTNLCAIHAKR). A ubiquitinated lysine at position 115 was identified. The Gly-Gly residue has a mass of 114.04 Da. The MS/MS spectra were obtained with HCD of the +4 m/z ion at 923.9716. y_i* = y_i − 57.02.