A Novel Nuclear Protein Complex Controlling the Expression of Developmentally Regulated Genes in Toxoplasma Gondii

Abstract

Toxoplasma gondii is a ubiquitous protozoan parasite with a complex life cycle containing multiple developmental stages. The parasites have distinct gene expression patterns at different stages to enable stage specific life activities, but the underlying regulatory mechanisms are largely unknown. In this study, a nuclear complex is identified that controls the expression of developmentally regulated genes. This complex consists of the AP2 family transcription factor AP2XII-5, the epigenetic factors MORC and HDAC3, as well as a novel AP2XII-5 interacting protein 1 (AIP1) that stabilizes this complex. At the tachyzoite stage when the parasites proliferate rapidly by asexual endodyogeny, AP2XII-5 binds to the promoter regions of developmentally activated genes and recruits MORC and HDAC3 to suppress their expression. When sexual commitment and merozoite development are triggered, the abundance of AP2XII-5 decreases and its suppression on target genes is relieved. In contrast to MORC and HDAC3, which regulate Toxoplasma development but are also essential for tachyzoite growth, AP2XII-5 and AIP1 are dispensable for tachyzoite proliferation in vitro. These data suggest that while MORC and HDAC3 have broad regulatory activities, forming a complex with AP2XII-5 and AIP1 enables them to specifically regulate gene expression during development.

Keywords: AP2 transcription factor; HDAC3; bradyzoite; epigenetic factor; merozoite.

Figure 1

TgAP2XII‐5 is dispensable for tachyzoite growth in vitro. a) Schematic representation of knocking out TgAP2XII‐5 by CRISPR/Cas9 assisted homologous gene replacement. Red bar indicates the CRISPR targeting site. b, c) Diagnostic PCRs and Western blotting on a representative clone of ΔXII‐5. d) Plaque assay comparing the overall growth of indicated strains under normal tachyzoite growth conditions. e, f) The parasitophorous vacuole (PV) size (e) and intracellular replication rates (f) of indicated strains under alkaline growth conditions (pH = 8.2, ambient CO₂). (e) Median with interquartile range of more than 100 plaques, student's t‐test. (f) Means ± SEM of n = 3 independent experiments, each with three replicates, two‐way ANOVA with Tukey's multiple comparisons post‐tests.

Figure 2

TgAP2XII‐5 inactivation leads to altered expression of a large number of developmentally regulated genes. a) Volcano plot showing differentially expressed genes in WT and ΔXII‐5 strains, as determined by RNA‐seq. Data from three biological replicates were plotted. Red and blue dots indicate the number of genes that were significantly up‐ and down‐regulated (p values < 0.01 and fold change (FC) ≥ 2) in the ΔXII‐5 mutant, respectively. b) Venn diagram illustrating the overlap between the genes silenced by AP2XII‐5 and those upregulated two‐fold or more in bradyzoites (BZ), merozoites (MZ), or sporozoites (SP) compared to tachyzoites (TZ) (data from ToxoDB). c) Western blot showing the protein expression of merozoite specific gene TgPAN in the WT and ΔXII‐5 parasites. TgPAN was detected by an HA antibody in the ME49/pan‐HA or ΔXII‐5/pan‐HA transgenic strains.

Figure 3

Identification of proteins interacting with TgAP2XII‐5. a) Proteins interacting with TgAP2XII‐5 were immunoprecipitated by an HA antibody from the lysate of the TgAP2XII‐5‐HA strain and identified by mass spectrometry. Co‐immunoprecipitation (co‐IP) using the ME49 strain was used as a control. The number of unique peptides for each identified protein in the co‐IP experiments was used to generate the plot. b) Selected proteins co‐precipitated with TgAP2XII‐5‐HA, HDAC3 or AIP1‐HA, as determined by mass spectrometry. The number of unique peptides for each hit in the experimental (exp) and control (ctrl, using untagged strains or negative serum for co‐IP) groups was indicated. A full list of hits was shown in Supplementary Data 1. c) Interactions between AP2XII‐5 and HDAC3 or MORC determined by co‐IP and Western blotting, using the TgAP2XII‐5‐HA strain. ME49 strain was used as a control. d) Interactions between AIP1 and AP2XII‐5‐Ty or MORC determined by co‐IP and Western blotting, using the AIP1‐mAID/XII‐5‐Ty strain.

Figure 4

TgAIP1 suppresses the expression of developmentally activated genes in tachyzoites. a) Volcano plot of differentially expressed genes before (‐IAA) and after (+IAA, 24 hours) AIP1 depletion in the AIP1‐mAID (iAIP1) strain, as determined by RNA‐seq analyses. Data from three biological replicates were plotted. Genes significantly up‐ or down‐regulated were defined as p < 0.01 and fold change (FC) ≥ 2. b) Venn diagram illustrating the overlap between genes silenced by TgAIP1 and the genes that are upregulated at different life cycle stages compared to tachyzoites (Source: ToxoDB), as done in Figure 2b. c) Venn diagram illustrating the overlap of genes suppressed by TgAIP1 and TgAP2XII‐5.

Figure 5

TgAIP1 stabilizes the AP2XII‐5‐MORC complex to suppress the expression of target genes. a, b) Protein levels of AP2XII‐5 and MORC in the iAIP1/XII‐5‐Ty strain treated with or without IAA. c, d) Protein interactions between AP2XII‐5 and MORC in the iAIP1/XII‐5‐Ty strain with or without IAA treatment, as determined by coIP and Western blotting. M: protein marker. e) Expression changes of selected genes in the indicated strains treated with or without IAA, as determined by RT‐qPCR. TgME49_235390 and TgME49_219828 are merozoite specific genes, while TgME49_294600 and TgME49_311710 are sporozoite specific genes. Beta tubulin was used as a normalization control. Means ± SEM of n = 3 independent experiments, unpaired two‐tailed student's t‐test. **P < 0.01.

Figure 6

AP2XII‐5 recruits HDAC3 to repress the expression of target genes. a) Binding sites of HDAC3 on the genomes of ME49 and the ΔXII‐5 strains, as determined by ChIP‐Seq. Chromosome Ib was shown as an example. The y‐axis depicts the abundance of ChIP‐Seq reads. b) AP2XII‐5 dependent recruitment of HDAC3 to the upstream regions of genes whose transcription was upregulated after AP2XII‐5 deletion. Binding of HDAC3 to the ‐2 kb to +2 kb regions of the transcription start sites (TSS) of target genes in the ME49 and the ΔXII‐5 strain was plotted. Data from n = 2 replicates of each strain were included. c) Binding of HDAC3 to the promoter regions of representative merozoite specific genes (GRA11B, TGME49_273985, and TgGRA80) in an AP2XII‐5 dependent manner, as determined by ChIP‐Seq. d‐e) Artificial delivery of HDAC3 to the promoter region of PAN in the ΔXII‐5 strain reduced PAN expression. HDAC3 was fused to dCas9‐GFP and expressed from a plasmid that also expressed a PAN targeting sgRNA (sgPAN). The plasmid (or the dCas9‐GFP empty vector as a negative control) was introduced into the ΔXII‐5 strain and IFA analysis was performed using an anti‐HA antibody to probe the expression of pan‐HA. Representative images taken with the same exposure time to compare the fluorescence intensity of pan‐HA were shown in (d). Quantification of the fluorescence intensity of pan‐HA in n ≥ 30 transfectants (indicated by GFP⁺ in the nuclei) was plotted in (e). Median with interquartile range of three independent experiments, unpaired two‐tailed Student's t‐test.

Figure 7

AP2XII‐5 is crucial for parasite virulence in vivo. a) Survival curves of KM mice infected with different strains. The infection dose is 100 parasites per mouse and each strain was tested with 10 mice. b) Number of cysts in the brains of mice that survived in (a). Means ± SEM of n = 3 mice from each group were analyzed, unpaired two‐tailed Student's t‐test. c) Dose dependent virulence of the ΔXII‐5 mutant. KM mice were injected with indicated doses of ME49 versus ΔXII‐5 tachyzoites and the survival of mice was monitored for 30 days. Each dose was tested by n = 10 mice.

Figure 8

A model for the role of the AP2XII‐5 containing complex in controlling the expression of developmentally regulated genes. a) AP2XII‐5 forms a complex with AIP1 and the epigenetic factors MORC and HDAC3 to silence the expression of target genes at the tachyzoite stage. When the parasites are stimulated to differentiate, the abundance of the AP2XII‐5 protein was decreased by an unknown mechanism, so that the repression of target genes was relieved. b) Western blotting checking the abundance of TgAP2XII‐5 in the iAP2XII‐1/AP2XII‐5‐Ty strain treated with IAA for different periods (0–72 h) that induced merogony.