A novel GCN5b lysine acetyltransferase complex associates with distinct transcription factors in the protozoan parasite Toxoplasma gondii

Abstract

Toxoplasma gondii is a protozoan parasite that has a tremendous impact on human health and livestock. High seroprevalence among humans and other animals is facilitated by the conversion of rapidly proliferating tachyzoites into latent bradyzoites that are housed in tissue cysts, which allow transmission through predation. Epigenetic mechanisms contribute to the regulation of gene expression events that are crucial in both tachyzoites as well as their development into bradyzoites. Acetylation of histones is one of the critical histone modifications that is linked to active gene transcription. Unlike most early-branching eukaryotes, Toxoplasma possesses two GCN5 homologues, one of which, GCN5b, is essential for parasite viability. Surprisingly, GCN5b does not associate with most of the well-conserved proteins found in the GCN5 complexes of other eukaryotes. Of particular note is that GCN5b interacts with multiple putative transcription factors that have plant-like DNA-binding domains denoted as AP2. To understand the function of GCN5b and its role(s) in epigenetic gene regulation of stage switching, we performed co-immunoprecipitation of GCN5b under normal and bradyzoite induction conditions. We report the greatest resolution of the GCN5b complex to date under these various culture conditions. Moreover, reciprocal co-IPs were performed with distinct GCN5b-interacting AP2 factors (AP2IX-7 and AP2XII-4) to delineate the interactomes of each putative transcription factor. Our findings suggest that GCN5b is associated with at least two distinct complexes that are characterized by two different pairs of AP2 factors, and implicate up to four AP2 proteins to be involved with GCN5b-mediated gene regulation.

Keywords: Acetylation; Apicomplexan; Epigenetics; Proteomics; Toxoplasma.

Figure 1.. Epitope tagging of TgAP2XII-4 at its endogenous locus.

A. Plasmid integration at the ap2xii-4 locus by single crossover recombination to insert sequence coding for a C-terminal 3xHA tag in frame with the ap2xii-4 coding sequence. B. Immunofluorescence staining of HA-tagged AP2XII-4^3xHA tachyzoites (red), co-localizing with DAPI DNA stain (blue). C. Western blotting of parental RHΔHXΔku80 and AP2XII-43xHA parasite lysates with anti-HA antibody.

Figure 2.. Schematic representation of the GCN5b interactome.

Overview of interacting proteins of GCN5b, AP2IX-7, and AP2XII-4 in tachyzoites under normal culture conditions. Target proteins that were immunoprecipitated are signified with thick borders. Interactors conserved in human and yeast SAGA complexes are colored blue. AP2 factors are green, proteins with an annotated function are in yellow, and hypothetical proteins are in grey. Blue shaded oval signifies the members of the AP2IX-7/AP2X-8 complex and the red shaded oval signifies the members of the AP2XII-4/AP2VIIa-5 complex.