Toxoplasma gondii AP2XII-2 Contributes to Transcriptional Repression for Sexual Commitment

Abstract

Toxoplasma gondii is a widespread protozoan parasite that has a significant impact on human and veterinary health. The parasite undergoes a complex life cycle involving multiple hosts and developmental stages. How Toxoplasma transitions between life cycle stages is poorly understood yet central to controlling transmission. Of particular neglect are the factors that contribute to its sexual development, which takes place exclusively in feline intestines. While epigenetic repressors have been shown to play an important role in silencing the spurious gene expression of sexually committed parasites, the specific factors that recruit this generalized machinery to the appropriate genes remain largely unexplored. Here, we establish that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci associated with sexually committed parasites along with epigenetic regulators of transcriptional silencing, HDAC3 and MORC. Despite its widespread association with gene promoters, AP2XII-2 is required for the silencing of relatively few genes. Using the CUT&Tag (cleavage under targets and tagmentation) methodology, we identify two major genes associated with sexual development downstream of AP2XII-2 control, AP2X-10 and the amino acid hydroxylase AAH1. Our findings show that AP2XII-2 is a key contributor to the gene regulatory pathways modulating Toxoplasma sexual development. IMPORTANCE Toxoplasma gondii is a parasite that undergoes its sexual stage exclusively in feline intestines, making cats a major source of transmission. A better understanding of the proteins controlling the parasite's life cycle stage transitions is needed for the development of new therapies aimed at treating toxoplasmosis and the transmission of the infection. Genes that regulate the sexual stages need to be turned on and off at the appropriate times, activities that are mediated by specific transcription factors that recruit general machinery to silence or activate gene expression. In this study, we identify a transcription factor called AP2XII-2 as being important for the repression of a subset of sexual stage genes, including a sexual stage-specific AP2 factor (AP2X-10) and a protein (AAH1) required to construct the infectious oocysts expelled from infected cats.

Keywords: Toxoplasma; apicomplexan parasites; gene expression; sexual development; transcription.

FIG 1

AP2XII-2 genome occupancy significantly overlaps those of HDAC3 and MORC. (A) AP2XII-2 genome occupancy along a segment of chromosome VIIa reveals its association with the promoters of several genes. Statistically enriched segments are shown as black bars directly under AP2XII-2-enriched peaks (purple). Similar distributions and enrichments of HDAC3 and MORC are seen in the orange and teal tracks, respectively. (B) Genome-wide analysis reveals that each member of the transcriptional repression complex associates with the transcriptional start sites (TSS) of genes. (C) Venn diagram indicating that the majority of the AP2XII-2-enriched peaks fall within those identified for HDAC3 and MORC. (D) Venn diagram indicating that the majority of genes occupied by AP2XII-2 are also occupied by HDAC3 and MORC. (E) Analysis of genes flagged as being bound only by AP2XII-2 in panel D. Genes are grouped into subsets of genes whose AP2XII-2 peaks overlap HDAC3/MORC peaks but were affected by gene annotation issues, genes associated with low peak scores, and genes that appear to be uniquely bound by AP2XII-2.

FIG 2

AP2XII-2 depletion leads to relatively little gene dysregulation. (A) Western blotting confirming the IAA-dependent downregulation of AP2XII-2^AID-HA protein in RH^TIR1 or ME49^TIR1 parasites after 24 h of incubation with 500 μM IAA in culture medium. Blots were probed with anti-SAG1 as a loading control. (B and C) MA plots of differential gene expression of AP2XII-2^AID-HA depleted in RH^TIR1 (B) or ME49^TIR1 (C) parasites 24 h after IAA addition. Significantly upregulated genes are in blue, and downregulated genes are in red. Genes of interest for further analysis are noted. (D) Venn diagrams showing the overlap of significantly upregulated and downregulated genes in the ME49 and RH genetic backgrounds. (E) Analysis of AP2XII-2 gene occupancy and directionality of dysregulation that occurs in the ME49 strain upon AP2XII-2 depletion. (F) Meta cell cycle expression analysis of significantly upregulated (blue) and downregulated (red) gene sets that respond to AP2XII-2 depletion. The cell cycle profile showing Growth 1 (G1), Synthesis (S), and Mitosis/Cytokinesis (M/C) phases of AP2XII-2 is shown in black as a reference.

FIG 3

Disentanglement of the aromatic amino acid hydroxylase family of genes on chromosome V. (A) Genomic alignment and syntenic analysis of RH88 and ME49 genomes available at ToxoDB.org reveal the correct positioning of the unplaced contigs under GenBank accession numbers KE139705 and KE139818 into chromosome V, which was missed due to the incorrect fusion of the AAH1/2 genes in the ME49 reference strain. Suggested changes to the annotations of AAH1 and AAH2 are based on sequence alignments of the 3′-flanking regions in both genes across both parasite strains. (B) Multiple pairwise alignments of CDS regions that are present in each AAH gene fragment reveal a high degree of identity at the nucleotide level, which obfuscates gene calling during differential gene expression analysis. (C) Pairwise alignment of the 3′-flanking regions reveals a clear way to discriminate both the AAH1 and AAH2 genes for C-terminal tagging at endogenous loci. (D) Outline of AAH family gene fragments available in the ME49 reference strain. Missing fragments at either the 5′ or the 3′ terminus are indicated. Conserved CDS regions that were used for multiple-sequence alignment are delimited by dotted lines.

FIG 4

Depletion of AP2XII-2 causes derepression of AAH1 gene expression. (A and B) Western blotting demonstrating that AAH1 (A) and not AAH2 (B) is expressed upon IAA-induced depletion of AP2XII-2. Both genes were endogenously tagged at their C termini with MYC epitope tags. Blots were probed with anti-SAG1 as a loading control. (C) AAH1 expression is observed in a concentric pattern around the nucleus upon AP2XII-2 depletion. (D) AAH1 is expressed in the vicinity of the parasite mitochondrial marker F₁B ATPase.