In Toxoplasma gondii, the expression of stage-specific genes is regulated by Apetala-2 (AP2) transcription factors. Recently, two TgAP2 transcription factors, restricted to tachyzoites, were shown to inhibit the induction of merogony: conditional depletion of these TgAP2s in tachyzoites induced the expression of merozoite-specific mRNAs and resulted in the emergence of merozoite phenotypes in vitro. Taking a different approach, we asked whether inducible ectopic expression of enteroepithelial stage (EES)-specific TgAP2s in tachyzoites, which are restricted to EESs, would result in the upregulation of substrate gene mRNAs of stage-regulated factors linked to merozoites, gamonts, and oocysts, with maximal mRNA levels in these developmental stages. In connection with experiments addressing this question, we are also exploring the role of epigenetic blocks by the MORC-HDAC complex, which restrict access of induced TgAP2s to binding sites of EES-specific substrate genes in tachyzoites. Therefore, we have re-designed the original experimental strategy to include the inhibition of histone deacetylase 3 (HDAC3). We will present RNA-Seq data showing the initial substrate gene pools of EES-specific TgAP2s, preliminary results of ChIP-Seq experiments as well as computational strategies for validating target sequences.

In summary, we present a refined model system to study gene regulation in cat enteroepithelial stages of T. gondii using a simple standard culturing method, eliminating the need for either experimental cat infections or specialized and labour-intensive cell culture systems such as enteroid cultures, and yielding robust and reproducible outputs.