GDV1 triggers sexual conversion and differentiation in malaria parasites by antagonizing HP1-dependent gene silencing

Malaria is caused by Plasmodium parasites that proliferate through iterative cycles of intra-erythrocytic replication. During each cycle a small number of parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)-dependent silencing of ap2-g prevents sexual conversion and secures proliferation. Here, we identify gametocyte development 1 (GDV1) as the first upstream activator of the sexual differentiation pathway in P. falciparum. Induction of GDV1 expression is sufficient to activate AP2-G expression and sexual differentiation. We found that GDV1 targets heterochromatin and triggers HP1 eviction preferentially at ap2-g and other gametocyte-specific genes. We further demonstrate that GDV1-dependent activation of ap2-g is controlled via a gdv1 antisense RNA. In summary, we identify GDV1 as an unprecedented cell fate decision factor that induces sexual differentiation by antagonizing HP1-dependent gene silencing. ChIP-seq experiments were performed for the gametocyte development protein 1 (PfGDV1, PF3D7_0935400) in the malaria parasite Plasmodium falciparum strain 3D7. An inducible ectopic overexpression 3D7/PfGDV1-GFP-DD transgenic parasite line was created using the FKBP-destabilization domain Shield-1 system on an episomal expression plasmid. The resulting PfGDV1-GFP-DD transgenic parasite line was used in a comparative ChIP-sequencing time-course experiment. 3D7/GDV1-GFP-DDOFF parasites were split at 28-34 hours post-invasion (hpi), cultured in parallel in absence or presence of Shield-1 and paired chromatin samples were harvested two (TP1), six (TP2) and ten (TP3) hours after Shield-1 addition. Chromatin immunoprecipitations were performed using mouse anti-GFP (Roche Diagnostics, #11814460001). In parallel the effect of PfGDV1 overexpression on global PfHP1 occupancy was investigated ten hours (TP3) after Shield-1 addition using anti-rabbit PfHP1 (Brancucci et al. 2014). Additionally, directional RNA-seq was performed for Plasmodium falciparum F12 wildtype parasites and a gdv1-antisense RNA loss-of-function mutant parasite line (F12/gdv1-asKO) generated using CRISPR/Cas9-based genome editing. F12 and F12/gdv1-asKO parasites were synchronized to obtain an eight hour growth window and were harvested at 32-40hpi and 40-48hpi.