Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii

Abstract

Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.

FIG.1.

Histone acetylation is a mark of gene activation during stage conversion. (A) Tachyzoite (T)-to-bradyzoite (B) conversion of Toxoplasma. The Prugniaud strain was induced in vitro with an alkaline pH. The kinetics of stage conversion was monitored by immunofluorescence assay with bradyzoite-specific antibodies (α-CC2 and α-BRS4) and tachyzoite-specific antibody α-SAG1. Note that in vitro conversion to bradyzoites is not 100% efficient, so our converted parasites are labeled B^/T to denote parasite populations greatly enriched for bradyzoites (70 to 80%). (B) Stage-specific and constitutively expressed genes examined by ChIP analysis. Genes: BAG, bradyzoite antigen; BSR, bradyzoite-specific recombinant; LDH, lactate dehydrogenase; SAG, surface antigen; SRS, SAG-related sequences; DEOC, deoxyribose-phosphate aldolase; GRA, dense granule protein; eIF4A, eukaryotic translation initiation factor 4A; DHFR, dihydrofolate reductase-thymidylate synthase. (C) ChIP analysis of housekeeping genes (eIF4A and DHFR), tachyzoite-specific genes (SAG1 and SAG2A), and bradyzoite-specific genes (DEOC, LDH2, and BAG1). Tachyzoites (T) and in vitro-generated bradyzoites (B^/T) were processed for ChIP analysis with α-acetyl [K9-K14] histone H3 and α-acetyl [K5-K8-K12-K16] histone H4 and α-histone H3 antibodies. A chromatin sample with an irrelevant antibody was used as a negative control (IgG) and genomic DNA as a positive control for PCR (input). The input PCR signal was set at 100%, and the numerical value of the ChIP signal represents the percentage of the input. The amount of immunoprecipitated DNA determined by semiquantitative PCR was normalized to the respective input DNA for each sample (shown in arbitrary units). Each bar is an average of three independent experiments. (D) Long-range ChIP analysis of the BSR4 locus. (E) ChIP analysis of the GRA gene family (GRA1 to GRA9).

FIG. 2.

Chromatographic purification of TgCRC, a TgHDAC3-containing complex. (A) Recombinant protein in transgenic parasites expressing TgHDAC3-HA-FLAG was detected by IFA with HA antibody and compared to nuclear localization of acetylated histone H4 (α-AcH4). (B) Purification scheme of the TgHDAC3-HA-FLAG complex. WCE from tachyzoites was fractionated by chromatography as described in Materials and Methods. The 0.35 M KCl elution of DEAE-Sephacel was purified with an anti-FLAG M2 affinity column. The bound proteins were further analyzed by Superose 6 gel filtration. The horizontal and diagonal lines indicate stepwise and gradient elutions, respectively. Molar concentrations are given. FT, flowthrough. (C) Silver staining and Western blotting (α-HA) of Superose 6 fractions (15 μl). Molecular weight markers are indicated on the left. (D) Superose 6 fraction 20 was trichloroacetic acid precipitated, separated by SDS-polyacrylamide gel electrophoresis (4% to 12%), and visualized by colloidal blue staining. Molecular weight markers are indicated on the left. The proteins identified by mass spectrometry sequencing are indicated. Polypeptides marked with asterisks are contaminants. (E) Structural features of TgCRC-100/TgTBL1, TgCRC-230, and TgCRC-350. aa, amino acids.

FIG.3.

Repurification of TgCRC with TgTBL1 as bait and determination of enzymatic activity. (A) Transiently expressed recombinant protein HA-FLAG-TgTBL1 was detected by IFA with an HA antibody and compared to nuclear localization of acetylated histone H4 (α-AcH4). (B) Purification scheme of TgTBL1-associated proteins. FT, flowthrough. (C) Silver staining of affinity-purified TgTBL1-containing complex compared to TgCRC (Superose 6, fraction 20). The proteins analyzed are indicated on the right. Western blot analysis using HA antibody detects tagged TgTBL1. WCE from untransfected wild-type RH tachyzoites was processed similarly as a control (mock). Molecular weight markers are indicated on the left. (D) HDAC activity assays of FLAG affinity-purified TgTBL1 or TgHDAC3. TSA, trichostatin A; APAH, aroyl-pyrrole-hydroxyamides.

FIG. 4.

ChIP analysis of TgHDAC3 and TgGCN5 binding sites. Tachyzoites from stable transgenic parasite clones expressing either FLAG-TgGCN5 or TgHDAC3-HA-FLAG were processed for ChIP with αα-FLAG antibody. Stage-specific promoters that were tested included SAG2A and an unknown EST (tachyzoite specific), as well as BAG1 and SAG2C (bradyzoite specific). A chromatin sample with an irrelevant antibody was used as a negative control (IgG) and genomic DNA as a positive control for PCR (input).

FIG.5.

Arginine methylation of Toxoplasma histones. (A) Immunofluorescence colocalization of methylated [R17] H3 and Hoechst 33258 within the nuclei of intracellular tachyzoites. (B) Evolutionary relationships between arginine methyltransferases of T. gondii TgCARM1 and TgPRMT1, Plasmodium falciparum PfCARM1 (accession no. CAD51260) and PfPRMT1 (accession no. AAN36855), Cryptosporidium parvum CpCARM1 (genomic annotation), Arabidopsis thaliana AtCARM1 (accession no. AAF26997), D. melanogaster DmCARM1 (CARMER, accession no. AAF54471) and DmPRMT1 (accession no. AAM11369), C. elegans CePRMT1 (accession no. T26447), S. cerevisiae ScHMT1 (accession no. NP_009590), and Homo sapiens HsCARM1 (accession no. AAH46240) and HsPRMT1 (accession no. AAF62895). The unrooted phylogenetic tree was inferred from the core domain alignment. (C) Recombinant histidine-tagged proteins (rTgCARM1 and rTgPRMT1) were expressed in E. coli and purified by Ni-nitrilotriacetic acid affinity chromatography. Methylation of H3 [R17] is mediated in vitro by rTgCARM1 (0.25 μg). The assays with recombinant H3 (1.5 μg) and free core histones (3 μg) were performed by immunoblotting with α-methyl [R17] H3 antibody. Methylation of H4 [R3] is mediated in vitro by rTgPRMT1 (2.5 μg). The assays with wild-type GST-histone TgH4 (1.5 μg), mutant GST-histone TgH4 (R3K, 1.5 μg), and free core histones (3 μg) were performed by immunoblotting with α-methyl [R3] H4 antibody. (D) Immunofluorescence assay with α-HA of transgenic parasite clone A3, engineered to ectopically express HA-FLAG-TgCARM1. (E) WCE of 5 × 10¹⁰ tachyzoites expressing HA-FLAG-TgCARM1 was fractionated with an anti-FLAG M2 affinity column. Bound proteins were further analyzed by chromatography on a Superose 6 column. Silver staining analysis of the anti-FLAG affinity eluates corresponding to fractions 17 to 41 was done. Western blotting was performed with the antibodies indicated on the left. W.T, wild type. (F) Methylation of nucleosomal histones (3 μg) by rTgCARM1 (0.25 μg) supplemented with 5 μg of Toxoplasma protein extract enriched in ATP-dependent nucleosome disruption activity. The concentration of ATP was 1.0 mM where added. The reaction mixtures were resolved by SDS-polyacrylamide gel electrophoresis, and [³H]histone H3 was excised for counting in scintillation buffer.

FIG. 6.

Concurrent histone acetylation and methylation in the epigenetic control of stage conversion. (A) ChIP analysis of tachyzoite-specific genes (unknown tachyzoite EST, SAG1, and SAG2A) and the bradyzoite-specific gene SAG2C. Tachyzoites (T) and in vitro-generated bradyzoites (Bradyzoite^/Tachyzoite) were processed for ChIP with TgCARM1 antibody (α-49), α-methyl [R17] H3, α-acetyl [K18] H3, or α-histone H3 antibodies. A chromatin sample with an irrelevant antibody was used as a negative control (IgG) and genomic DNA as a positive control for PCR (input). (B) Yeast (Sc) and Toxoplasma (Tg) GCN5 acetylation of recombinant histone H3 (1 μg) in vitro. Substrate specificity was monitored by immunoblotting HAT assay reaction mixtures with antibodies to specific acetylated (Ac) lysine residues.

FIG. 7.

AMI-1 inhibits TgCARM1 activity in vitro and induces cyst formation in vivo. (A) In vitro inhibition by AMI-1 and activation by AMA-2 of methylation reactions mediated by rTgCARM1 (0.25 μg) and rTgPRMT1 (0.2 μg). Methylation reactions were performed with histone H3 (1.5 μg), wild-type GST-histone TgH4 (1.5 μg), and free core histone substrates (3 μg) in the presence of dimethyl sulfoxide (DMSO), AMI-1 (50, 200, and 600 μM), AMA-2 (50, 200, and 600 μM), and S-adenosylhomocysteine (SAH; 0.04, 0.16, and 0.32 μM). PW, DNA molecular weight. (B) Estimation of AMI-1-dependent in vitro conversion. The ratio of parasites that were reactive either with tachyzoite-specific α-SAG1 antibody or bradyzoite-specific α-CC2 antibody was determined by double immunofluorescence as described in Materials and Methods. Mab, monoclonal antibody. (C) Phase-contrast microscopy and immunofluorescence assays with AMI-1-treated tachyzoites with α-methyl [R17] H3 and α-CC2 antibodies. The decrease of methylation at arginine 17 localizes within the induced bradyzoites as assessed by α-CC2 immunofluorescence. (D) Immunofluorescence of in vitro-generated bradyzoites induced by an alkaline pH with α-methyl [R17] H3 and α-CC2 antibodies. (E) ChIP experiments with extracellular tachyzoites treated with AMI-1 or dimethyl sulfoxide (control) display the binding of methylated H3 [R17] on TgCARM1 target SAG1 and SAG2A genes (Fig. 6A). The n-fold enrichment was calculated by dividing the α-methyl [R17] H3 intensity signal by the input control signal. The data from five independent experiments were pooled in order to graph the average enrichment. The primers used in the PCR analysis were designed to span the region showing the highest peak of enrichment for each promoter (region A, Fig. 6A). (F) Model of epigenetic control of stage-specific gene expression in T. gondii.