Regulation of the developmental programs in Toxoplasma by a novel SNF2L-containing chromatin remodeling complex

Abstract

Toxoplasma gondii is an extremely successful parasite infecting one third of the human population and numerous animals. It has a complex life cycle with multiple developmental stages that are key for its transmission and pathogenesis. But how the developmental programs are regulated is largely unknown. Here, we screen putative chromatin remodeling proteins in T. gondii and find that a novel complex containing an evolutionarily conserved ATPase SNF2L is critical for programming the parasite's development. This complex contains four core proteins and conditional depletion of three of them leads to similar expression changes of developmentally regulated genes, including increased transcription of genes involved in sexual commitment and development. Accordingly, depletion of SNF2L causes merogony and out-budding types of division, which are otherwise only observed at the enteroepithelial stages within definitive hosts where sexual reproduction of the parasite occurs. After being recruited to target regions, SNF2L regulates gene expression by modulating local chromatin accessibility or by recruiting accessory proteins to its binding sites, thus ensuring that the gene expression and reproduction patterns are matched to the life cycle stages. Conditional depletion of SNF2L offers an opportunity to study the unique biology of the parasite during pre-sexual and sexual developments in vitro.

Fig. 1. Putative chromatin remodeling ATPases in Toxoplasma.

a Domain structures of the putative chromatin remodeling ATPases in Toxoplasma identified by BLAST searches. DEXDc: DEAD-like helicases superfamily (SMART #: SM00487); HELICc: helicase superfamily c-terminal domain (SMART #: SM00490); SANT: SANT SWI3, ADA2, N-CoR and TFIIIB” DNA-binding domains (SMART #: SM00717); AT_hook domain: DNA binding domain with preference for A/T rich regions (SMART # SM000384); SnAC: Snf2-ATP coupling, chromatin remodeling complex (SMART #: SM01314); zf-PARP: Poly(ADP-ribose) polymerase and DNA-Ligase Zn-finger region (SMART #: SM01336); RING: Ring finger (SMART #: SM00184); UBA_4: UBA-like domain (Pfam #: PF14555); COM: Chromatin organization modifier domain (SMART #: SM000298); DUF: DUF4208 domain (SMART #: SM001176); PHD: PHD zinc finger (SMART #: SM000249); BROMO: bromo domain (SMART #: SM000297); SNF2_N: SNF2_N domain (Pfam #: PF00176). b Phylogenetic analyses of the putative chromatin remodeling ATPases in Toxoplasma, which were done in MEGA 11 using the maximum likelihood algorithm. c IFA checking the localization of putative chromatin remodeling ATPases in Toxoplasma, as well as the depletion of their expression by IAA treatments, using strains with each of the ATPases tagged with an mAID degron (which contains an HA tag) at their C-termini in the endogenous gene loci. d Impact of chromatin remodeling ATPases depletion on parasite growth, as determined by plaque assays using mAID tagged strains with or without IAA treatment. The parental strain ME49-Tir1 was included as a control. e Impact of the depletion of chromatin remodeling ATPases on parasite division. All patterns that are different from the regular endodyogeny are classified as “Abnormal”. Means ± SD of n = 3 independent experiments, ****p < 0.0001, two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.

Fig. 2. SNF2L depletion leads to altered modes of parasite reproduction.

a Western blotting demonstrating the depletion of SNF2L expression by IAA treatment in the SNF2L-mAID (iSNF2L) strain. ALD was included as a loading control. b Intracellular replication assay illustrating the reduced growth rates and chaotic division patterns of the SNF2L depletion mutants. The number of parasites in each PV of the iSNF2L and ME49-Tir1 strains treated with or without IAA for 24 h (with 12 h of ± IAA pretreatment before the replication assay) was counted. Means ± SEM of n = 3 independent experiments, ***p < 0.0001, Kolmogorov-Smirnov test. SNF2L depletion leads to altered modes of parasite reproduction, as revealed by IFA staining of IMC1, IMC7, or GAP45 in the iSNF2L/IMC7-Myc (c) or iSNF2L (d) strains. The +IAA parasites in (d) were treated with IAA for 36 h in total (12 h of pretreatment in T25 flasks followed by 24 h of treatment on coverslips before imaging). Arrowheads in (c) indicate parasites with replicating/segregating nuclei but no daughter cell formation, whereas arrows indicating endopolygeny-like division. Yellow arrows in (d) indicate splitting of mother parasite or its nucleus, whereas arrowheads indicate parasites without nuclear DNA. e Percentage of SNF2L-depleted parasites undergoing indicated types of reproduction described in (d). More than 100 vacuoles with identifiable mode of reproduction were analyzed in n = 3 independent experiments. Data are presented as means ± SD. Source data are provided as a Source Data file.

Fig. 3. Out-budding in SNF2L depleted mutants.

a endodyogeny of the iSNF2L strain without IAA treatment. Each mother cell contained two daughter cells (D), and each daughter cell had a single nucleus (Nu). b–g reproduction patterns and morphologies of the iSNF2L strain treated with IAA, which displayed out-budding (bud) type of division. In addition, some parasites contained multiple mitochondria (Mi). Source data are provided as a Source Data file.

Fig. 4. SNF2L depletion results in expression changes of genes involved in parasite development.

a Venn diagram showing the overlap of genes upregulated (≥2-fold) in the SNF2L-depleted mutants with the genes that are upregulated in wildtype merozoites (Mz), bradyzoites (Bz), or sporozoites (Sp) compared with tachyzoites (Tz). Data for the latter are from published datasets available in ToxoDB. ***p < 0.0001, Pearson’s Chi-squared tests indicating genes upregulated in the SNF2L depletion mutant were significantly enriched in gene sets associated with bradyzoites, merozoites, or sporozoites. b For the 301 genes that were upregulated in the SNF2L-depleted mutants, their expression patters in wildtype strain during the life cycle were plotted as a heatmap using data from ToxoDB. c Life cycle expression patterns of the top 30 upregulated genes (excluding hypothetical proteins) in the SNF2L-depleted mutants were plotted as a heatmap. d expression patterns HAP2, PF16, and TGME49_30633839 during the parasites’ life cycle, as well as in iSNF2L mutants with or without IAA treatment. e Volcano plot showing the abundance change of proteins in the iSNF2L strain before and after IAA treatment for 12 h, as determined by TMT-based quantitative proteomics. Data from three biological replicates were plotted. Raw data are shown in Supplementary data 3. f Expression of PF16 in the SNF2L-depleted mutant, as determined by IFA on the iSNF2L/PF16-Ty strain with or without IAA treatment for 60 h. Source data are provided as a Source Data file.

Fig. 5. SNF2L binds to the promoter regions of target genes and its depletion alters chromatin accessibility.

a Enrichment of SNF2L near the transcription start site (TSS) (from −3 Kb to 3 Kb region centered around TSS), as determined by CUT&Tag in the SNF2L-3HA strain using an HA antibody. Pull-down with a naive mouse IgG was included as a control. b Genome browser view of the position and intensity of DNA sequences bound by SNF2L. Data were derived from (a) and similar CUT&Tag experiments done in the iSNF2L strain with or without 12 h IAA treatment. Chromosome XII was shown as an example. c Distribution of SNF2L on different types of genetic elements, derived from the analysis of data in (a). d Enrichment of SNF2L at the upstream regions of selected genes, using data from (a). Meanwhile, the accessibility of chromatin revealed by ATAC-Seq at these regions in the iSNF2L strain treated with or without IAA was also included for comparison. e, f Overall chromatin accessibility in the iSNF2L strain treated with or without IAA, as determined by ATAC-Seq (e). Chromosome XII was shown as an example (f). g accessibility of different types of genetic elements in the iSNF2L strain with and without IAA treatment, derived from the analysis of data in (e). h accessibility of the upstream regions of genes that are upregulated (upper panel) or downregulated (lower panel) in the SNF2L-depleted parasites. Means ± SEM were plotted. Source data are provided as a Source Data file.

Fig. 6. SNF2L interacts with novel nuclear factors to form a gene regulation complex.

a–d Protein hits from co-IP experiments that were used to identify binding partners for the prey proteins (marked in red). The number of unique peptides derived from untagged (Ctrl) and HA tagged (HA) strains was used to generate these graphs. e co-IP results analyzed by the Straightforward Filtering IndeX (SFINX) to show the possible protein interactions in the SNF2L-containing complex. Raw data are shown in Supplementary data 5. f–h co-IP and Western blotting examining the interactions between SNF2L, SLIF1, and SLIF2. i Cartoons showing the SNF2L/ISW2 chromatin remodeling complexes in indicated organisms. j–l Heat maps showing the life cycle expression patterns of genes upregulated (≥2-fold) in SLIF1, SLIF2, or AP2X-4 depleted mutants. Data were derived from ToxoDB. m Venn diagram showing the overlap of genes upregulated (≥2-fold) in SNF2L, AP2X-4, and SLIF1 depleted mutants. Source data are provided as a Source Data file.