A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum

Abstract

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 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 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 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.

Fig 1. Identification of the PfGCN5 Core Complex in P. falciparum.

(A) Proteins identified from parasite nuclear extracts by IP and by LC-MS/MS. TAP procedure was performed using the PfGCN5::PTP line at the trophozoite stage (three replicates R1 –R3), while single-step IPs with the anti-Myc and anti-GFP beads were done using the PfPHD1::Myc parasite line at the trophozoite stage and PfGCN5::GFP at the ring stage (two replicates, R1, and R2). The wildtype 3D7 was used as IP control. The wildtype 3D7 was used as IP control. The proteomic data were analyzed by SAINT using a threshold of probability >94% and 1% FDR. Nine proteins consistently identified are marked as the PfGCN5 complex core subunits. Gene ID and annotation are shown on the right. (B) Schematic diagrams showing the features (putative domains and protein size) of the core subunits. HAT: histone acetyltransferase enzymatic domain; ADA2: ADA2 binding domain; BrD: Bromodomain; Med15: Med15 domain; ADA2LD: ASA2-like domain; PHD-SF: PHD finger superfamily; ePHD: extended PHD finger; PHD-TAF3: PHD finger homologous to transcription initiation factor TFIID subunit 3 (TAF3); AT: AT hook; TM: transmembrane region; AP2: AP2-domain; cyclin-like: cyclin-like domain; NAP: nucleosome assembly protein. The C-terminals of PfGCN5 and PfPHD1 labeled by the blue line and “X” containing BrD and PHD-TAF3 were deleted for functional studies (see details in S6 Fig). (C) Gel filtration analysis of the PfGCN5 complex. Aliquots of different fractions were used for Western blots with anti-PTP, PfPHD1 and PfPHD2 antibodies to detect full-length of PfGCN5, PfPHD1 and PfPHD2, and for the HAT assay using recombinant histone H3.

Fig 2. Growth phenotypes in PfGCN5 and PfPHD1 domain deletion mutants.

(A) Asexual growth rates of WT 3D7, PfGCN5-ΔBrD::GFP, and PfPHD1-ΔPHD::GFP. ** indicate P < 0.01 (ANOVA) at days 5 and 7. The starting parasitemia was 0.1%. (B) The distribution of the number of mature schizonts with a variable number of merozoite. No differences were identified among the three parasite lines (P > 0.05, ANOVA). (C) Invasion rates of the GCN5-ΔBrD::GFP and PHD1-ΔPHD::GFP parasite lines estimated by incubating purified schizonts with RBCs (P < 0.05, paired Wilcoxon test). (D) The duration of the IDC showing significantly increased lengths in GCN5-ΔBrD::GFP and PHD1-ΔPHD::GFP parasite lines (P < 0.05, paired Wilcoxon test). (E, F) Detailed analysis of the IDC showing extended ring stage in the GCN5-ΔBrD::GFP (E) and PHD1-ΔPHD::GFP (F). (G) Gametocytemias at day 6 after induction of gametocytogenesis showing significantly increased gametocytemia in the two domain deletion mutants (P < 0.05, paired Wilcoxon test).

Fig 3. Domain deletions affect the abundance and localization of active histone marks and the integrity of the PfGCN5 complex.

(A) The levels of active histone marks in 3D7, PfGCN5-ΔBrD::GFP (ΔBrD), and PfPHD1-ΔPHD::GFP (ΔPHD) parasite lines. Histones were purified from the ring, trophozoite, and schizont stages, and detected by Western blots with specific antibodies against the modified histones H3K9ac, H3K14ac, H3K4me3, and H4Acs. Anti-H3 antibodies were used for loading control. The Western blots were performed in three biological replicates, and the band intensities were determined using a densitometer. The number underneath each band indicates the fold change ± standard deviation between the ratio of the corresponding histone mark normalized to the H3 control. The ratios in 3D7 at the ring stage were set as 1. (B, C) Co-localization of full-length PfGCN5 (GCN5::GFP) (B) or truncated PfGCN5 (GCN5-ΔBrd::GFP) (C) with H3K9ac and DAPI by IFA with anti-GFP and H3K9Ac antibodies. Note the expansion of the truncated PfGCN5-ΔBrD::GFP and H3K9ac beyond the periphery of the euchromatin areas demarcated by DAPI staining. (D) Effects of domain deletions in PfGCN5 and PfPHD1 on complex integrity. Proteins were pulled down from the trophozoite nuclear extracts of the PfGCN5::GFP, GCN5-ΔBrD::GFP, and PHD1-ΔPHD::GFP parasite lines and identified by LC-MS/MS. R1, R2, R3 and R4 indicate individual repeats of the experiment. Shown here are proteins passing the threshold of SAINT (probability >94% and FDR <1%). The nine PfGCN5 complex core subunits were all detected in the IPs of PfGCN5::GFP and GCN5-ΔBrD::GFP, whereas only four of the core subunits were identified in the IPs of PHD1-ΔPHD::GFP.

Fig 4. Global transcriptomic changes up domain deletions in PfGCN5 and PfPHD1.

(A) The phaseograms of transcriptome from the WT 3D7, PfGCN5-ΔBrD::GFP (ΔBrd), PfPHD1-ΔPHD::GFP (ΔPHD) showing the disturbance of the cascade-like gene expression pattern in the deletion mutants at different developmental stages. R, ring; ET, early trophozoite; LT, late trophozoite; S, schizont. (B, C) Volcano plots showing altered gene expression at the ring (B) and schizont (C) stages in PfGCN5-ΔBrD::GFP compared to the WT 3D7. The x-axis indicates log₂ (Fold change) of the transcript level in PfGCN5-ΔBrD::GFP compared to WT 3D7, while the y-axis indicates -log₁₀ of the P values.(D) Number of genes with altered expression at different developmental stages of the IDC in the two domain deletion mutants. The up- and down- regulated genes are labeled in red and blue, respectively. (E, F) Volcano plots showing altered gene expression at the ring (E) and schizont (F) stages in PfPHD1-ΔPHD::GFP compared to the WT 3D7. The x-axis indicates log₂ (Fold change) of the transcript level in PfPHD1-ΔPHD::GFP compared to WT 3D7, while the y-axis indicates log₁₀ of the P values. (G, H) Overlaps of down-regulated (G) and up-regulated (H) genes between the PfGCN5-ΔBrD::GFP and PfPHD1-ΔPHD::GFP parasite lines at different stages. (I, J) The enriched functions or pathways of up-regulated and down-regulated genes upon PfGCN5 BrD (I) and PfPHD PHD deletion determined by GSEA. Each pixel represents the normalized enrichment score (NES). The positive and negative NESs represent up-and down-regulated functions after domain deletions, respectively.

Fig 5. Virulence gene expression altered upon domain deletions.

(A) The expression of total var genes at different developmental stages. The y-axis indicates the total reads of var genes from RNA-seq. R, ring; ET, early trophozoite; LT, late trophozoite; S, schizont. (B) Pie graphs showing the overall levels of the var gene transcripts in the WT 3D7, PfGCN5-ΔBrD::GFP, and PfPHD1-ΔPHD::GFP parasite lines at the early trophozoite stage. The numbers in parentheses are the total expression of all var genes identified by RNA-seq analysis. (C) Western blot showing PfEMP1 protein levels in the iRBC membranes of the WT 3D7, PfGCN5-ΔBrD::GFP, and PfPHD1-ΔPHD::GFP parasite lines with the anti-ATS antibodies. Aldolase (ALD) expression was used as the loading control. (D) Bar graph showing the overall PfEMP1 protein levels in WT 3D7, PfGCN5-ΔBrD::GFP, and PfPHD1-ΔPHD::GFP parasite lines. Bars indicate means and standard deviations from five replicates. (E) Representative images of RNA FISH analysis showing the single locus of the B-type var gene expression in the WT 3D7 and more than one B-type var gene locus in the two deletion mutants.

Fig 6. Down-regulation of invasion-related pathway and alteration of AP2 genes in domain deletion mutants.

(A) Heatmaps displaying down-regulation of genes involved in the invasion of the RBC in the two deletion mutants. * and # indicate the AP2-I and putative AP2-LT target genes, respectively. Ø and Ψ indicate the down-regulated genes upon knockdown of BDP1 and the target genes identified by BDP1-HA ChIP-seq analysis, respectively. (B) Heatmaps showing altered expression of the AP2 genes in the deletion mutants. (C) Putative AP2-LT target genes (with AP2-LT binding motifs) are significantly enriched in the down-regulated genes in PfGCN5-ΔBrD::GFP at the late stages. The numbers of up- and down-regulated genes upon PfGCN5 BrD domain deletion are shown as the red and blue bars at four developmental stages. These altered genes were further classified as the putative target genes of AP2-LT and the remaining genes. R, ring; ET, early trophozoite; LT, late trophozoite; S, schizont. ***, P <0.001 (Fisher’s exact test).

Fig 7. Correlation of genes showing altered expression in domain deletion mutants with promoter accessibility and chromatin states.

(A, B) Changed levels of gene expression in PfGCN5-ΔBrd and PfPHD1-ΔPHD are negatively correlated with the accessibility of the promoters (from the ATAC-seq analysis, The y-axis shows that the ratio of ATAC-seq peak (scaled per million reads) vs genomic DNA library) (A), but are positively correlated with the heterochromatin state (represented by the HP1 occupancy) (B). U, upregulation; D, downregulation. *, P <0.05; ****, P < 0.0001 (Wilcoxon rank-sum test). (C) Heatmaps displaying the transcriptional activation of gametocyte and ookinete genes in the domain deletion mutants. R, ring; ET, early trophozoite; LT, late trophozoite; S, schizont. (D) Overlaps of activated gametocyte- or ookinete-specific genes between PfGCN5-ΔBrd and PfPHD1-ΔPHD.