The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex inPlasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasite lines with either the bromodomain in PfGCN5 or the PHD domain in PfPHD1 deleted. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific down-regulation of merozoite genes involved in erythrocyte invasion, many of which contain the AP2-LT binding motif and are also regulated by AP2-I and BDP1, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entirevargene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silent and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including an AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as ‘Sinton and Mulligan’ stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum.

Protein arginine methyltransferases (PRMTs) regulate many important cellular processes, such as transcription and RNA processing in model organisms but their functions in human malaria parasites are not elucidated. Here, we characterize PfPRMT5 in Plasmodium falciparum, which catalyzes symmetric dimethylation of histone H3 at R2 (H3R2me2s) and R8, and histone H4 at R3 in vitro. PfPRMT5 disruption results in asexual stage growth defects primarily due to lower invasion efficiency of the merozoites. Transcriptomic analysis reveals down-regulation of many transcripts related to invasion upon PfPRMT5 disruption, in agreement with H3R2me2s being an active chromatin mark. Genome-wide chromatin profiling detects extensive H3R2me2s marking of genes of different cellular processes, including invasion-related genes in wildtype parasites and PfPRMT5 disruption leads to the depletion of H3R2me2s. Interactome studies identify the association of PfPRMT5 with invasion-related transcriptional regulators such as AP2-I, BDP1, and GCN5. Furthermore, PfPRMT5 is associated with the RNA splicing machinery, and PfPRMT5 disruption caused substantial anomalies in RNA splicing events, including those for invasion-related genes. In summary, PfPRMT5 is critical for regulating parasite invasion and RNA splicing in this early-branching eukaryote.

During development within the host erythrocyte malaria parasites generate nascent membranous structures which serve as a pathway for parasite protein transport to modify the host cell. The molecular basis of such membranous structures is not well understood, particularly for malaria parasites other than Plasmodium falciparum. To characterize the structural basis of protein trafficking in the Plasmodium knowlesi-infected erythrocyte, we identified a P. knowlesi ortholog of MAHRP2, a marker of the tether structure that connects membranous structures in the P. falciparum-infected erythrocyte. We show that PkMAHRP2 localizes on amorphous structures that connect Sinton Mulligan's clefts (SMC) to each other and to the erythrocyte membrane. Three dimensional reconstruction of the P. knowlesi-infected erythrocyte revealed that the SMC is a plate-like structure with swollen ends, reminiscent of the morphology of the Golgi apparatus. The PkMAHRP2-localized amorphous structures are possibly functionally equivalent to P. falciparum tether structure. These findings suggest a conservation in the ultrastructure of protein trafficking between P. falciparum and P. knowlesi.

The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum , which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasites with the bromodomain deletion in PfGCN5 and the PHD domain deletion in PfPHD1. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific downregulation of merozoite genes involved in erythrocyte invasion, many of which harbor the DNA-binding motifs for AP2-LT and/or AP2-I, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific transcription factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silenct and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including the AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist. Author Summary: Epigenetic regulation of gene expression plays essential roles in orchestrating the general and parasite-specific cellular pathways in the malaria parasite Plasmodium falciparum . Using tandem affinity purification and proteomic characterization, we identified a divergent transcription co-activator – the histone acetyltransferase GCN5-associated complex in P. falciparum , which contains nine core components, including two PHD domain proteins (PfPHD1 and PfPHD2) and a plant apetela2-domain transcription factor. To understand the functions of the PfGCN5 complex, we performed gene disruption in two subunits of this complex, PfGCN5 and PfPHD1. We found that the two deletion mutants displayed very similar growth phenotypes, including significantly reduced merozoite invasion rates and elevated sexual conversion. These two mutants were associated with dramatic decreases in histone H3K9 acetylation and H3K4 trimethylation, which led to global changes in chromatin states and gene expression. Genes significantly affected by the PfGCN5 and PfPHD1 gene disruption include those participating in parasite-specific pathways such as invasion, virulence, and sexual development. In conclusion, this study presents a new model of the PfGCN5 complex for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.