The G9a histone methyltransferase inhibitor BIX-01294 modulates gene expression during gametocyte development and transmission

Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is initiated by sexual precursor cells, the gametocytes. Gametocytes are formed in response to stress signals and following up-take by a blood-feeding Anopheles mosquito initiate sexual reproduction. Gametocytes need to fine tune their gene expression in order to develop inside the mosquito to continue life-cycle progression. Previously we showed that post-translational histone acetylation controls gene expression during gametocyte development and transmission. However, the role of histone methylation remains poorly understood. We here use the histone G9a methyltransferase inhibitor BIX-01294 to investigate the role of histone methylation in regulating gene expression in gametocytes. Growth assays demonstrated that BIX-01294 inhibits intraerythrocytic replication with an IC50 of 13.0 nM. Furthermore, BIX-01294 significantly impairs gametocyte maturation and reduces the formation of gametes and zygotes. Comparative transcriptomics between BIX-01294-treated and untreated immature, mature and activated gametocytes demonstrated greater than 1.5-fold deregulation of over 359 genes. The majority of these genes are transcriptionally down-regulated in the activated gametocytes and could be assigned to transcription, translation, and signaling indicating a contribution of histone methylations in mediating gametogenesis. Our combined data shows that inhibitors of histone methylation may serve as a multi-stage antimalarials. Plasmodium falciparum Percoll enriched immature (stages II-IV) gametocytes (imGC), mature (stage V) gametocytes (mGC), and gametocytes at 1 h post-activation with 100 µM Xanthurenic acid at RT (aGC) were obtained. Each enriched gametocyte sample was treated with BIX-01294 at IC90 concentrations (0.12 µM) or with 0.5% vol. DMSO (untreated control) for 1 h and 6 h, respectively and total RNA was isolated using the Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The extracted RNA was then processed for microarray analysis.