PROJECT SUMMARY Epigenetics machinery has been used as important therapeutic drug targets evident by the successful inhibitors for cancer treatment. Although the malaria parasite harbors many epigenetic regulators, it remains a great challenge to discover unique ones diverse from those in the human host. Based on previous studies and our recent preliminary data, PfSET7, one of ten SET-domain containing lysine (K) methyltransferases (KMTs) in Plasmodium falciparum, is discovered to be a Plasmodium-specific KMT with unique structures and functions. PfSET7 does not belong to any known SET family in model organisms because it contains a unique motif II in the SET domain. PfSET7 was found localized at the apical tip of merozoite and downregulation of PfSET7 by ~35% using our newly developed CRISPR interference (CRISPRi) resulted in defects in merozoite egress and invasion, reminiscent of the findings that Toxoplasma gondii apical complex KMT (TgAKMT) is also localized at the apical tip before egress and disruption of TgAKMT led to defects in parasite egress and invasion due to impaired motility. However, TgAKMT’s mode of action, especially its substrates, is still unknown. Remarkably, PfSET7 was found also localized in the gametocyte cytoplasm and nucleus, and downregulation of PfSET7 by ~26% also led to defects in gametocyte development, coincidentally with the findings that PfSET7 has peak expression in gametocytes and many distinct methylated lysines were identified in gametocyte histones. Notably, PfSET7 contains four Plasmodium-specific regions and is only ~41% identical to TgAKMT, indicating PfSET7 harbors malaria-specific structures. Based on these findings, we hypothesize that PfSET7 is a Plasmodium-specific KMT and has a dual function in the regulation of gametocyte gene expression and merozoite motility during egress/invasion by targeting histone and non-histone proteins, respectively. To decipher the critical function and substrate specificity of PfSET7, we will apply advanced new technologies including a stronger CRISPRi KD with optimized gRNAs and a conditional knockout for gene functional study, and quantitative methylome for substrate identification. It is anticipated that this study will lay a solid foundation for understanding PfSET7’s mechanism of action and the development of novel apicomplexan- specific epigenetic drugs.

PROJECT NARRATIVE Discovering unique epigenetic targets in the human malaria parasite Plasmodium falciparum with function and structure diverse from those in the human host is critical for developing novel interventions in malaria control. Based on previous studies and our preliminary data, we propose to define the function and substrates of PfSET7, a SET-domain containing lysine methyltransferase with unique structures and functions in two pivotal blood stages: merozoite for invasion and gametocyte for transmission. With the completion of this project, we will reveal the unique function and specificity of PfSET7 in P. falciparum and will lay a solid foundation for the development of novel apicomplexan-specific epigenetic drugs.