The malaria parasite Plasmodium falciparum employs antigenic variation of the virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) to escape adaptive immune responses during blood infection. Antigenic variation of PfEMP1 occurs through transcriptional switches in the mutually exclusive expression of individual members of the multi-copy var gene family. var genes are located in perinuclear clusters of transcriptionally inactive heterochromatin. Singular var gene activation is linked to locus repositioning into a dedicated zone at the nuclear periphery and deposition of histone 3 lysine 4 di-/trimethylation (H3K4me2/3) and H3K9 acetylation marks in the promoter region. While previous work identified the putative H3K4-specific methyltransferase PfSET10 as an essential enzyme and positive regulator of var gene expression, a recent study reported conflicting data. Here, we used iterative genome editing to engineer a conditional PfSET10 knockout line tailored to study the function of PfSET10 in var gene regulation. We demonstrate that PfSET10 is not required for mutually exclusive var gene expression and switching. We also show that PfSET10 is dispensable not only for asexual parasite proliferation but also for sexual conversion and differentiation of gametocytes. Furthermore, comparative RNA-seq experiments revealed that PfSET10 plays no obvious role in regulating gene expression during asexual parasite development and gametocytogenesis. Interestingly, however, PfSET10 shows different subnuclear localization patterns in asexual and sexual stage parasites and female-specific expression in mature gametocytes. In summary, our work confirms in detail that PfSET10 is not involved in regulating var gene expression and is not required for blood stage parasite viability, at least not when cultured in vitro, thus suggesting PfSET10 may be important for life cycle progression in the mosquito vector or during liver stage development.
Overall design: Synchronized ring stage parasites (0-6 hpi) cultured in the presence of 2.5 ng/μl BSD-S-HCl were split into three 10 ml cultures. Dish 1 (BSD control, baseline reference) was maintained under 2.5 ng/μl BSD-S-HCl selection. Dishes 2 (DMSO control, PfSET10 WT) and 3 (RAPA-treated, PfSET10 KO) were released from BSD-S-HCl pressure and treated with either the DMSO solvent alone or 100 nM RAPA, respectively. Dishes 1-3 were all cultured in parallel for two additional generations to allow for parasite expansion, and sorbitol synchronizations were performed to secure synchronous parasite populations for sample harvest. At the early ring stage (4-10 hpi), the DMSO- and RAPA-treated cultures were split into four equal 10 ml cultures each to be able to harvest four time points across the IDC (TP1, 10-16 hpi; TP2, 22-28 hpi; TP3, 30-36 hpi, TP4, 38-44 hpi), and one separate 10 ml dish each was maintained in culture for ten additional generations. The BSD control ring stage sample (R-WT-G0) was harvested alongside the DMSO- and RAPA-treated ring stage samples (R-WT-G2, R-KO-G2) (TP1), and trophozoite, early schizont and late schizont samples of the DMSO-treated (T-WT-G2, ES-WT-G2, LS-WT-G2) and RAPA-treated (T-KO-G2, ES-KO-G2, LS-KO-G2) cultures were harvested at the corresponding TPs 2-4 in the same IDC. For the DMSO- and RAPA-treated cultures, an additional 10-16 hpi ring stage sample each was harvested ten generations after release from BSD-S-HCl pressure (R-WT-G10, R-KO-G10). For gametocyte samples, DMSO- and RAPA-treatment was performed on synchronous ring stage parasite cultures (0-8 hpi). After two additional invasion cycles, 20 ml parasite culture each was induced using mFA as explained above and the ring stage progeny was split into two 10 ml dishes, cultured in the presence of 50 mM GlcNAc for five days and total RNA samples were harvested on day 6 (D6-WT, D6-KO) and day 12 of gametocytogenesis (D12-WT, D12-KO), respectively. All samples have been harvetsed in biological triplicates.
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