Toxoplasma H2A variants reveal novel insights into nucleosome composition and functions for this histone family

Abstract

Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here, we report the characterization of three H2A histones, variants H2AX and H2AZ, and a canonical H2A1. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)varphi (where varphi denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We found that expression of h2ax, but not h2a1 or h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are expressed but h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this stage of the life cycle. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation.

Figure 1. Sequence alignment and phylogenetic analysis of histones H2A

(a) Alignment of Toxoplasma H2A1 (Gene IDs at www.toxodb.org: 55.m04926; TGME49_061250; TGGT1_chrVIIb 1811408–1811794 and TGVEG_chrVIIb 1800384–1800743), H2AX (Gene IDs at www.toxodb.org: 55.m04942, TGME49_061580, TGGT1_008650 and TGVEG_073130) and H2AZ (Gene IDs at www.toxodb.org: 145.m00002, TGME49_100200, TGGT1_095100 and TGVEG_007580) amino acid sequences. Predicted sites of ubiquitylation (Ub), α-helix structure and loops L1 and L2 are indicated. (b) Neighbor joining (NJ) tree of H2A amino acid sequences. Box: H2AZ subfamily. At, Arabidopsis thaliana (Accession numbers: AAL85051 and BAF00012), Dm, Drosophila melanogaster (Accession numbers: AAN11125 and AAF56631), Gl, Giardia lamblia (Accession number: AF139873); Hu, human (Accession numbers: AAN59958, P16104 and CAG33696), Pf, Plasmodium falciparum (Accession numbers: P40282 and CAB39069); Pyy, Plasmodium yoelii yoelii (Accession numbers: CAQ40903 and EAA15833); Sc, Saccharomyces cerevisiae (P04909, P04910 and Q12692), Tg, Toxoplasma gondii, Tt, Tetrahymena thermophilus (AAC37292, AAC37291 and CAA33554), Xl, Xenopus laevis. Accession numbers (AAA49769, AAH74188 and AAH74203)

Figure 2. Sequence analysis of histones H2A

(a) Comparison of the deduced amino acid sequences of histones H2A N-terminal region. Tg: T. gondii histones. Pf: P. falciparum histones Hu: human histones (Accession numbers: AAN59958, P16104 and CAG33696). Sc: S. cerevisiae histones (P04909, P04910 and Q12692). HTA1 and HTA2 are H2AX histones; HTAZ1 is H2AZ variant. The panel shows sites of post-translational modifications identified in human, yeast and Plasmodium H2A. Post-translationally modified residues are indicated as shaded letters. Cylinder indicates part of the histone core. Histone A repressive domain (HAR) is underlined.

Figure 3. Generation of recombinant histones and specific antibodies

(a) The specificity of the antibodies generated was tested based on their ability to recognize the recombinant Toxoplasma histones by Western blot. rH2A1, rH2AX and rH2AZ were used to study α-H2A antibodies (α-H2A1 L1,α-H2A1 L2, α-H2AX, α-H2AZ and α-H2AZNt); rH2Ba and rH2Bv were used to detect cross-reaction of α-H2Bv antibody. (b) IFA of intracellular RH tachyzoites performed with α-H2A1 L1, α-H2AX, α-H2AZ and antibodies, and DAPI (nuclear labeling). hN = host cell nucleus. (c) Toxoplasma histones analyzed by Western blot with α-H2AZNt, α-H2AX and α-H2A1 L1 antibodies. Histones (H) were purified by acid extraction from RH tachyzoites, electrophoresed in 15% SDS-PAGE, and blotted onto nitrocellulose. Filters were stained with Ponceau red and each one was cut in two pieces. They were then analyzed by Western blot (WB). In both cases, the other H2As were compared to α-H2AX bands. In the blot localized at the left is α-H2AZ Nt whereas in the one at the right side is α-H2A1 L1.

Figure 4. Histone – histone interactions

(a) Tachyzoites were permeabilized with digitonin and treated with micrococcal endonuclease (MNase) in order to obtain mononucleosomes-containing solution to perform co-immunoprecipitations. The presence of mononucleosomes was evaluated by isolating DNA from an aliquot and examining it on a 1.5% agarose gel. (b) Lysates 1, 2 and 3 (L1, L2 and L3) were used to perform immunoprecipitations (IP) with α-H2AX, α-H2AZ and α-H2Bv, respectively. The input (IN) corresponds to 1 % of each lysate. Immunoprecipitated material was then examined by Western blotting (WB) for the presence of H2AZ, H2AX, H2Bv and SAG1 (as a contamination control).

Figure 5. Association of Toxoplasma H2A and H2Bs with active chromatin

Tachyzoite lysates generated by sonication were used for co-IP analysis. (a) Antibodies against Toxoplasma H2AZ, H2AX, H2Bv were used to perform co-IP. They were analyzed by Western blots with the same antibodies to ensure the IP worked correctly (CTR). Additionally, anti-acetylated H3 (α-AcH3) antibody was used for Western blot. (b) IP was performed with anti-acetylated lysine (α-AcLys) antibody and Western blot analysis were performed with α-AcH3 (positive control), α- H2A1 L1, α-H2AX, α-H2AZ, and α-H2Bv antibodies. IN: input (1% of tachyzoite whole extract).

Figure 6. Genomic distribution of H2A and H2B variants

(a) Chromatin immunoprecipitation (ChIP) was carried out using α-H2AX, α-H2AZ and α-H2Bv. In addition, α-H4K20me1 and α-AcH3 antibodies were used as heterochromatin and euchromatin controls, respectively. Immunoprecipitated DNA was then analyzed by qPCR with primers of sag1, β-tubulin (tub), bag1 and ldh2 promoters and two TgIRE regions. (b) Localization of TgIRE sequences on Toxoplasma chromosomes as detected by in silico analysis (white boxes). Numbers below chromosomes indicate the position of TgIRE in each chromosome, identified by roman numerals.

Figure 7. Phosphorylation and expression analysis of h2a genes following DNA damage

Tachyzoites were treated with 0, 100, 200, or 400 µM H₂O₂ for 1 h at 37°C. (a) Western blots were performed to detect phospho H2AX (α-γH2AX) at the left and total H2AX (α-H2AX) at the right. Each one was also blotted for tubulin (α-Tub) as a loading control. Band intensities were quantified and histones were related to tubulin intensities (Ratio). b) Expression of h2ax, h2a1 and h2az was analyzed by qRT-PCR normalized to β-tubulin and calibrated to the control without H₂O₂. This graphic represents one of three independent experiments.

Figure 8. h2a gene expression and bradyzoite induction

(a) and (b). RHΔuprt parasites were induced to bradyzoites using pH 8.1 and low CO₂ for 4 days. Relative quantification of expression levels of several genes in tachyzoites (Tz) and in vitro bradyzoites (Bz) by qRT-PCR calibrated to tachyzoites using β-tubulin as endogenous control. (a) Efficiency of bradyzoite induction (higher than 80%) was analyzed by qRT-PCR of bradyzoite-specific genes bag1 and ldh2 and by IFA using Dolichos biflorus lectin (DBL) staining (cyst wall labeling) and anti-BAG1 antibody (labels the bradyzoite cytosol). (b) Expression of histones H2A. Log₂ of fold change Bz to Tz are graphed for each gene analyzed. Three independent experiments were performed and significant differences between Tz and Bz for each gene amplified were analyzed by Student T test (GraphPad Prism software) * p<0.05; *** p<0.001. Tachyzoite-specific gene sag1 was used as a control. (c) Table summarizing microarray data for select histone genes expressed in RH parasites stressed for 3 days with pH 8.1 relative to parallel culture maintained in normal pH. 1.0 indicates no change between stressed and unstressed parasites. (d) Brain cysts (BC) were purified from mice brains and visualized by phase contrast microscopy. They were processed to obtain mRNA of mature bradyzoites used to analyze H2A expression by reverse transcription and PCR assay.