Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation

Abstract

Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged dominant-negative form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). Proteomics studies revealed that GCN5b interacts with AP2-domain proteins, apicomplexan plant-like transcription factors, as well as a "core complex" that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.

Figure 1. Inducible expression of ectopic _ddHAGCN5b and _ddHAGCN5b(E703G) in Toxoplasma.

Immunofluorescence assays (IFAs) and Western blots using an anti-HA antibody on (A) _ddHAGCN5b and (B) _ddHAGCN5b(E703G) parasites, cultured in the presence (+) or absence (−) of 500 nM Shield for 48 hours. Anti-HA signal is shown in green and nucleic acid staining with DAPI is depicted in blue for the IFAs. Scale bar = 2 µm. Antibodies against β–tubulin were used as a loading control on the Western blots.

Figure 2. Induced expression of _ddHAGCN5b(E703G) arrests parasite replication.

Doubling assays were performed to assess growth of (A) parental wild-type, (B) _ddHAGCN5b, and (C) _ddHAGCN5b(E703G) parasites. Intracellular parasites were physically released from host cells and an equal number of parasites were allowed to infect fresh HFF monolayers. The infected cultures were subjected to the indicated Shield concentrations for 48 hr. Parasite proliferation was monitored by quantifying the numbers of parasites in 50 random vacuoles.

Figure 3. Reduced histone H3 acetylation in _ddHAGCN5b(E703G) parasites.

_ddHAGCN5b and _ddHAGCN5b(E703G) parasites were cultured in the presence of 500 nM Shield for 48 hr. Equivalent amounts of parasite lysate obtained from Shield- vs. vehicle-treated parasites were analyzed by Western blotting using antibodies recognizing various acetylated lysine (K) residues of histone H3 (acetylated K9, K14, and K18). Anti-HA was used to monitor protein stabilization. Antibodies to total H3 show that induced stabilization of each GCN5b protein did not alter overall H3 protein levels. Western analysis of β-tubulin levels served as an additional loading control. Densitometry was used to compare relative levels of each histone acetylation mark between the Shield- and vehicle-treated parasites normalized to β-tubulin.

Figure 4. KDAC inhibitors (KDACi) restore replication of dominant-negative GCN5b parasites.

_ddHAGCN5b(E703G) tachyzoites were treated with a range of concentrations of either TSA or apicidin in combination with 500 nM Shield-1. Infected cells with no KDACi were included as a control. The number of parasite plaques in each infected host cell monolayer was counted after 7 days. Representative images of infected wells are depicted underneath the bar for each treatment. Each sample was performed in triplicate, with error bars showing standard deviation. Data shown are from one representative experiment from 3 independent trials that produced similar results. Asterisks denote statistical significance according to two-tailed student t-test; *** p<0.005, **p<0.01, *p<0.05.

Figure 5. GCN5b associates with genes containing introns, but is not preferentially associated with promoters.

The ChIP-chip results for TgME49_202650 are shown. The top tracing shows the H3K9ac ChIP-chip result (purple), which indicates the location of active promoters in Toxoplasma. Underneath are the individual results for each of the three ChIP-chip replicates (see Supplemental Data S1) plotted as the log2 ratio between the experimental GCN5b ChIP and input DNA (scale bar on the right). High stringency peaks with FDR<0.05 are shown in red. Peaks with 0.05<FDR<0.1 are shown in orange; 0.1<FDR<0.2 are shown in yellow; and peaks with 0.2<FDR<1 in gray. Only high stringency peaks (FDR<0.05) were used for the statistical analysis performed in this study. The bottom panel displays the gene prediction and the positions of its introns and exons (ToxoDB.org).

Figure 6. Reciprocal immunoprecipitation confirms the in vivo interaction of GCN5b with endogenously HA-tagged AP2IX-7 and AP2X-8.

A. IFAs showing localization of each AP2 to the parasite nucleus. Anti-HA (green) shows localization of designate HA-tagged AP2 protein; DAPI (blue) co-stains the nuclei. B. Immunoprecipitations using an anti-HA antibody were performed on parasite lysates made from AP2IX-7_HA, AP2X-8_HA, and AP2X-5_HA parasites, as well as the parental RHΔku80 line. The immunoprecipitated complexes were analyzed by Western blot using antibodies recognizing anti-HA, GCN5b, or β-tubulin. Arrowheads designate the expected size of each tagged AP2 protein (these AP2 proteins are very large and various breakdown products were observed in the different conditions used to process lysates versus IPs).