A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment

Abstract

Toxoplasma gondii has a complex life cycle that is typified by asexual development that takes place in vertebrates, and sexual reproduction, which occurs exclusively in felids and is therefore less studied. The developmental transitions rely on changes in the patterns of gene expression, and recent studies have assigned roles for chromatin shapers, including histone modifications, in establishing specific epigenetic programs for each given stage. Here, we identified the T. gondii microrchidia (MORC) protein as an upstream transcriptional repressor of sexual commitment. MORC, in a complex with Apetala 2 (AP2) transcription factors, was shown to recruit the histone deacetylase HDAC3, thereby impeding the accessibility of chromatin at the genes that are exclusively expressed during sexual stages. We found that MORC-depleted cells underwent marked transcriptional changes, resulting in the expression of a specific repertoire of genes, and revealing a shift from asexual proliferation to sexual differentiation. MORC acts as a master regulator that directs the hierarchical expression of secondary AP2 transcription factors, and these transcription factors potentially contribute to the unidirectionality of the life cycle. Thus, MORC plays a cardinal role in the T. gondii life cycle, and its conditional depletion offers a method to study the sexual development of the parasite in vitro, and is proposed as an alternative to the requirement of T. gondii infections in cats.

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Fig. 1: MORC, a highly conserved ATPase protein, interacts with HDAC3 in T. gondii.
Fig. 2: Recruitment of HDAC3 to chromatin is mediated by MORC.
Fig. 3: Depletion of MORC phenocopies the inhibition of HDAC3 by inducing the expression of sexual-stage-specific genes.
Fig. 4: Degradation of MORC, similar to the inhibition of HDAC3, induces the expression of merozoite-restricted transcripts.
Fig. 5: MORC regulates developmental transitions at multiple checkpoints.
Fig. 6: MORC guides developmental trajectories recruiting downstream regulating pathways.

Data availability

The data supporting the findings of this study are available from the corresponding author on reasonable request. The RNA-seq demultiplexed FASTQ files and gene-wise quantifications have been deposited in the NCBI Gene Expression Omnibus (GEO) and are accessible under accession number GSE136123. The ChIP–seq data have been deposited in the GEO under accession number GSE136060. The MS proteomics data have been deposited in the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifiers PXD016846 (MORC interactome) and PXD016845 (proteome-wide analyses). Source data for Figs. 1, 2 and 5 are presented with this paper.

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Acknowledgements

We thank the developers of the ToxoDB.org Genome Resource. ToxoDB and EuPathDB are part of the National Institutes of Health/National Institutes of Allergy and Infectious Diseases (NIH/NIAID)-funded Bioinformatics Resource Center. We also thank G. Communie for his help computing Danpos software. This work was supported by the Laboratoire d’Excellence (LabEx) ParaFrap (grant no. ANR-11-LABX-0024), the Agence Nationale pour la Recherche (Project HostQuest, grant no. ANR-18-CE15-0023 and ProFI grant no. ANR-10-INSB-08-01), the European Research Council (ERC Consolidator grant no. 614880 Hosting TOXO to M.-A.H.) and Fondation pour la Recherche Médicale (FRM grant no. FDT201904008364 to D.C.F.).

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Contributions

M.-A.H. supervised the research. M.-A.H. and D.C.F. generated the genetic tools, performed and analysed all of the genome-wide studies (RNA-seq, ChIP–seq and MNase assay), conducted most of biochemical experiments and the immunofluorescence assays. C.D., D.C. and F.S. generated biochemical reagents. L.B. and P.-J.D.B. performed MS analysis. Y.C. guided the MS experiments. C.S. performed structural modelling and analysed the MNase assay. A.B. computed and analysed the RNA-seq data. P.O. and M.B. analysed the ChIP–seq data. M.-A.H. and D.C.F. wrote the paper.

Corresponding author

Correspondence to Mohamed-Ali Hakimi.

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Extended data

Extended Data Fig. 1 Domain architectures and expression levels during the life cycle of MORC and partners.

a, Representative domain architectures of T. gondii MORC and partners identified by mass spectrometry-based proteomic are shown. Domains were predicted by SMART and PFAM: ELM2 (Egl-27 and MTA1 homology 2), PHD (plant homeodomain), and AP2 (APETALA2) b, Heatmap representation of MORC and partners gene expression in different life cycle stages (source: ToxoDB). Gene expression values were mean log2 transformed and median centered for clustering. Transcriptomic data from tachyzoite, merozoite, longitudinal studies on enteroepithelial stages (EES1 to EES5), immature (day 0), maturing (day 4) and mature (day 10) stages of oocyst development and cysts from chronically infected mice were used. c, Mapping of domains and identified phosphorylation and ubiquitination sites detected by mass-spectrometry (source: ToxoDB).

Extended Data Fig. 2 mAID-based MORC inducible KD system successfully ablated its expression on the protein post-translational level in both type I (RH) and type II (Pru) strains.

a, Auxin-inducible degradation system for controlling protein stability in T. gondii. We first engineered RH and Pru strains ectopically expressing the plant auxin receptor called transport inhibitor response 1 (TIR1). We chose the UPRT locus to integrate TIR1 under the control of a promoter allowing a mild expression of the protein tagged to Ty. We utilized a mini-AID (mAID) tagging LIC system for conditional MORC depletion. The resulting cell lines are referred to as MORC-KD hereafter. Conditional depletion of MORC-mAID-HA is reliant on auxin (IAA), TIR1, and the proteasome. b, Depletion of MORC-mAID-HA, upon addition of IAA for 16 hours, was measured by IFA in cells infected with Pru MORC KD. Fixed and permeabilized parasites were probed with MORC-mAID-HA (red) and GAP45 (green). MORC-depleted cells displayed strong inhibition of proliferation when compared to untreated cells. Yet, this growth defect phenotype was completely reversed upon IAA washout, indicating that MORC depletion while impeding the cell cycle progression does not kill the parasites. Graph on the right, in situ quantification of nuclear MORC-mAID-HA using IFA. The horizontal bars represent the mean ± s.d. of the nuclear MORC intensity from three independent experiments (n = 50 nuclei per dot). The p-values were calculated using one-way ANOVA. Scale bar, 10 μm. c, in situ quantification (related to Fig. 2c) of nuclear MORC-mAID-HA using IFA in RH MORC KD as described above. d, Smoothed and background-subtracted tag density profiles are displayed over representative regions of Chr. V (top) and X (bottom). The ChIP-seq profiles were obtained with antibodies directed against pan-acetylated histone H4, HDAC3 and HA (MORC detection) from chromatin sampled from a Pru MORC KD strain left untreated (UT) and or treated with IAA for 24 hours. The experiment was repeated independently twice with similar results.

Extended Data Fig. 3 MORC protein depletion, and HDAC3 inhibition induces gene expression.

a, Volcano plots showing gene expression differences identified from comparison of UT versus FR235222 in Pru MORC KD (left graph) and of UT versus IAA (24 hours) in RH MORC KD (right graph) (n=8914 genes, Supplementary Table 3). The orange and green dots indicate the number of significantly up- and down-regulated genes, respectively, using adjusted p < 0.01 (Bonferroni-corrected) and ± 2-fold change as the cut-off threshold corresponding to each comparison. X-axis showing log2 fold change, Y-axis showing -log10(p value). b, Volcano plots illustrating changes in protein expression between UT versus FR235222 in Pru MORC KD (left graph, n=2566 proteins, Benjamini-Hochberg FDR = 1 %, p-value ≤ 0.00794) and of UT versus IAA (24 hours) in RH MORC KD (right graph, n=2139 proteins, Benjamini-Hochberg FDR = 1.01%, p-value ≤ 0.00501). Overexpressed proteins upon MORC depletion are indicated in red and under-expressed ones in blue. c, Pru strains were engineered to endogenously epitope tag with HA two MORC-regulated genes, TGME49_207210 and TGME49_216140. Expression was monitored following HDAC3 chemical inactivation by FR235222 and HA staining. Scale bars, 10 μm. Experiments were conducted more than three times and representative images are displayed.

Extended Data Fig. 4 MORC alongside HDAC3 represses the expression of sexual stages-specific genes.

a, A type II (ME49) strain was engineered to endogenously expressed a HA tag version of the merozoite gene TGME49_238915. Genetic inactivation of HDAC3, MORC and AP2-XII-1 promote TGME49_243940 protein expression (in red). The efficiency of genetic disruption in Cas9-expressing parasites was monitored by cas9-GFP expression (in green). Scale bar, 5 μm. b, Expression of TGME49_243940 was monitored following HDAC3 chemical inactivation by FR235222 and HA staining (in red). Scale bar, 10 μm. c, Heatmap showing hierarchical clustering analysis of selected MORC-regulated genes from cluster 1 through different strain/induction combinations, including the abundance of their transcripts in the various stages of development, namely tachyzoite, bradyzoite/cyst, merozoite, enteroepithelial stages (EES) and oocyst stages. The color scale bar indicates log2 fold changes. d, Expression of TGME49_316130 was monitored following HDAC3 chemical inactivation by FR235222 and HA staining (in red). Scale bar, 10 μm. Experiments in a, b and d were conducted more than three times and representative images are displayed.

Extended Data Fig. 5 MORC KD derepresses proteins involved in merozoite and fertilization.

a, Smoothed and background-subtracted tag density profiles are displayed over Chr. VI. The ChIP-seq profiles were obtained with antibodies directed against various histone marks, HDAC3 and HA (MORC detection) from chromatin sampled from a Pru MORC KD strain left untreated (UT) and or treated with IAA for 24 hours. RNA-seq data through different strain/induction combinations are shown in black. The y-axis depicts read density from ChIP-seq and RPKM values for RNA-seq data. The merozoite-specific gene TGME49_238915 is shown in dark blue. b, Pru strain was engineered to endogenously expressed HA epitope-tagged TGME49_238915. Expression was monitored following HDAC3 chemical inactivation by FR235222 and HA staining. Scale bar, 10 μm. Experiment was conducted more than three times and representative images are displayed. c, Density profiles are displayed on Chromosome V at the TgHAP2 (TGME49_285940) locus. d, Density profiles are displayed on Chromosome X around a merozoite-specific cluster of tandemly repeated genes (SRS48 family). (a, c-d) The experiment was repeated independently twice with similar results. Chromosomal positions are indicated on x-axis.

Extended Data Fig. 6 MORC regulates microgamete related genes including those coding for flagella components.

Heatmap representation of RNA-seq data portraying the number of genes involved in microgamete biology, alongside their levels of expression upon MORC depletion/HDAC3 inhibition through different strain/induction combinations. The abundance of their transcripts in the various stages of development, namely tachyzoite, bradyzoite/cyst, merozoite, enteroepithelial stages (EES) and oocyst stages was displayed. The color scale bar indicates log2 fold changes. The genes were divided in sets grouping together genes involved in axonemal cytoskeleton, and genes that harbor potential domains involved in intraflagellar transportation.

Extended Data Fig. 7 MORC depletion induces the expression of genes involved in oocyst wall formation and coding for sporozoite-specific markers.

a, Density profiles are displayed on Chromosome VIIb at the SporoSAG/SRS28 (TGME49_258550) locus, encoding for the hallmark surface antigen in sporozoite. The ChIP-seq profiles were obtained with antibodies directed against various histone PTMs, HDAC3 and HA (MORC detection) from chromatin sampled from a Pru MORC KD strain left UT and or treated with IAA for 24 hours. RNA-seq data through different strain/induction combinations are shown in black. The y-axis depicts read density from ChIP-seq and RPKM values for RNA-seq data. b, Density profiles are displayed on Chromosome XI at the TgHowp1 (TGME49_316890) locus. TgHOWP1 is a representative example of proteins expressed in the early stages of wall formation as revealed by the accumulation of its transcripts in both late developmental EES5 and in immature (D0) oocyst (ToxoDB data). c, Density profiles are displayed on Chromosome VIIb at the TGME49_262110 locus. Expression of TGME49_262110 was monitored following HDAC3 chemical inactivation by FR235222 and HA staining (in green). Experiments was conducted more than three times and representative images are displayed. Scale bar, 5 μm. (a-c) The experiment was repeated independently twice with similar results. Chromosomal positions are indicated on x-axis.

Extended Data Fig. 8 MORC depletion derepresses partly bradyzoite-specific genes.

a, Heatmap showing hierarchical clustering analysis of selected bradyzoite-specific and MORC-regulated genes from cluster 1 through different strain/induction combinations. The abundance of their transcripts in the various stages of development, namely tachyzoite, bradyzoite/cyst, merozoite, enteroepithelial stages (EES) and oocyst stages was displayed. The color scale bar indicates log2 fold changes. b, Density profiles are displayed on Chromosome VIIb at the Bag1 (TGME49_259020) locus, encoding for the hallmark surface antigen in bradyzoite. The ChIP-seq profiles were obtained with antibodies directed against various histone PTMs, HDAC3 and HA (MORC detection) from chromatin sampled from a Pru MORC KD strain left UT and or treated with IAA for 24 hours. RNA-seq data through different strain/induction combinations are shown in black. The y-axis depicts read density from ChIP-seq and RPKM values for RNA-seq data. The experiment was repeated independently twice with similar results. Chromosomal positions are indicated on x-axis.

Extended Data Fig. 9 MORC depletion causes indirect repression of tachyzoites specific genes.

a-b, Density profiles are displayed at the tachyzoite-specific ROP16 (a) and RON2 (b) genes. The ChIP-seq profiles were obtained with antibodies directed against various histone marks, HDAC3 and HA (MORC detection) from chromatin sampled from a Pru MORC KD strain left UT and or treated with IAA for 24 hours. RNA-seq data through different strain/induction combinations are shown in black. The y-axis depicts read density from ChIP-seq and RPKM values for RNA-seq data. The experiment was repeated independently twice with similar results. Chromosomal positions are indicated on x-axis.

Extended Data Fig. 10 MORC regulates the expression of AP2IV-3, a Plasmodium falciparum AP2-G homologous protein.

a, Density profiles are displayed at the merozoite-specific AP2IV-3 gene. The ChIP-seq profiles were obtained with antibodies directed against various histone marks, HDAC3 and HA (MORC detection) from chromatin sampled from a Pru MORC KD strain left UT and or treated with IAA for 24 hours. RNA-seq data through different strain/induction combinations are shown in black. The y-axis depicts read density from ChIP-seq and RPKM values for RNA-seq data. The experiment was repeated independently twice with similar results. Chromosomal positions are indicated on x-axis. b, Representative domain architectures of T. gondii AP2IV-3 and P. falciparum AP2-G, displaying the approximate AP2 domain position within the protein sequence. c, Protein alignment of the AP2IV-3 and AP2-G respective AP2 domains, with the amino acid homology shown in red.

Supplementary information

Supplementary Information

Supplementary discussion, references and Figs. 1–7, and legends for Supplementary Tables 1–5.

Reporting Summary

Supplementary Table 1

MS-based characterization of the MORC interactome. Proteins eluted from Flag-tagged MORC co-immunoprecipitations performed from the RH and Pru strains were identified using MS-based proteomics. Toxoplasma proteins reproducibly identified from both strains with at least two peptides are presented. The identities of the proteins (accession number on ToxoDB, gene name and description) are indicated in columns A, B and C. Their molecular weights (MW) are noted in column D. The number of peptides identified for each protein is given in columns E and F for the RH and Pru strains, respectively. The abundance ranks of each protein in each interactome were deduced from extracted intensity-based absolute quantification values and are presented in columns G and H for the RH and Pru strains, respectively.

Supplementary Table 2

Description of T. gondii strains, plasmids, DNA synthesis and primers. List of T. gondii parasite lines as well as the plasmids used in this study. The primers and DNA synthesis construct used in this work are also shown in the table.

Supplementary Table 3

MORC- and HDAC3-regulated transcriptomes. Gene expression profiles in HFF showing that T. gondii genes are differentially regulated after MORC depletion or HDAC3 inhibition through different strain/induction combinations. RPKM values are shown for the indicated samples. The mean values for each gene/condition are indicated as well the replicate (REP) values. Differences in gene expression were identified from a comparison of (1) Pruku80 MORC KD left untreated (UT) or treated with IAA (24 h or 48 h); (2) RHku80 MORC KD left untreated or treated with IAA (7 h or 24 h); and (3) Pruku80 left unstimulated or treated FR235222 (FR) for 7 h or 18 h. Gene name (column A) and ToxoDB description (column B) are indicated.

Supplementary Table 4

MORC-regulated and stage-specific gene subsets as clustered in the study. A list of T. gondii genes related to Fig. 3b (columns A, B, C and D), Supplementary Fig. 3 (columns E and F) and Fig. 3g,h (columns G, H, I and J).

Supplementary Table 5

MS-based proteome-wide analyses following MORC KD or HDAC3 inhibition by FR235222. The proteomes from type I or type II strains infecting HFF cells were analysed by label-free quantitative proteomics (n = 8,322 predicted proteins). The quantification of proteins was based on razor + unique peptides (column E). Statistical significance was tested using limma. Differentially abundant proteins (marked by ‘1’ in column I) were defined by a log2-transformed fold change of ≥ 0.8 or ≤ −0.8 (column G) and P values (column H) were allowed to reach an FDR of ~1% according to the Benjamini–Hochberg estimator. Sheet 1: quantitative analysis of proteomes from Pru MORC KD parasites and Pru untreated (UT) parasites. P value cut-off = 0.00501. The red cells (column I) correspond to proteins that were enriched in MORC KD parasites compared with untreated parasites, whereas the blue cells (column I) correspond to proteins that were enriched in untreated parasites compared with MORC KD parasites. Sheet 2: quantitative analysis of proteomes from RH MORC KD parasites and RH untreated (UT) parasites. P value cut-off = 0.00631. The red cells (column I) correspond to proteins that were enriched in MORC KD parasites compared with untreated parasites, whereas the blue cells (column I) correspond to proteins that were enriched in untreated parasites compared with MORC KD parasites. Sheet 3: quantitative analysis of proteomes from Pru parasites that were either treated or not treated with FR235222. P value cut-off = 0.00794. The red cells (column I) correspond to proteins that were enriched in FR235222-treated parasites compared with untreated parasites, whereas the blue cells (column I) correspond to proteins that were enriched in untreated parasites compared with FR235222-treated parasites.

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Farhat, D.C., Swale, C., Dard, C. et al. A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment. Nat Microbiol 5, 570–583 (2020). https://doi.org/10.1038/s41564-020-0674-4

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