Proteomic analysis of parasites expressing PfMORCGFP reveals PfMORC association with nuclear proteins of epigenetic regulation.

(A) Volcano plot illustrates the protein enrichment in lable free LC-MS/MS analysis of PfMORC CoIPed proteins from three independent experiments at 32hpi. For normalized MS/MS counts, student’s t-test was performed and proteins were ranked as -log2 fold-change (x-axis) versus statistical p-values (y-axis). Gray dashed horizontal line shows the p-value cutoff. (B) Comparative analysis showing the juxtaposition of specific proteins CoIPed in PfMORCGFP with selected proteins from recent works of Hillier et al., Bryant et al. and Subudhi et al. where ApiAP2 or ISW1 were used as bait in similar CoIP experiments. The Venn diagram illustrates the overlap between identified proteins, revealing that the intersecting proteins are primarily ApiAP2 and chromatin remodellers. (C) An interactive protein-protein interaction network is constructed with proteins known to interact with PfMORC, using proteins identified in this study and proteins documented in previously published works. Proteins identified in this study with known interaction networks from the STRING database were used to curate the network employing Cytoscape to enrich the network quality.

Potential PfMORC interacting proteins enriched in CoIP eluates and identified in LC‒MS/MS from three independent experiments and fold change ≥ 1.5x GFP/3D7.

Genome-wide occupancy of PfMORC reveals localization to hypervariable surface antigen genes at 30 h and 40 h.

(A) Coverage tracks of PfMORC across all 14 P. falciparum chromosomes. Plotted values are fold enrichment (Log2[IP/Input]) of a representative replicate at 30 h. (B) Zoom-in of the last 100kb region of chromosome two from Panel A. Gene annotations represented in blue bars (P. falciparum 3D7 strain, version 3, release 57; PlasmoDB.org). (C) Mean fold enrichment of PfMORC occupancy across all var genes (top left), all rif genes (top right), and all stevor genes (bottom right), excluding pseudogenes. Graphical representation of exons to scale for each gene family annotated below enrichment plot in grey (e1=exon one; e2=exon two). (D) Quantitative Venn diagram comparing the number of MACS2 called peaks across each timepoint (light pink for 30 h; dark pink for 40 h). (E) Pie charts showing the type of genomic locations PfMORC peaks overlap at both 30 h and 40 h. Pink slices are 5’ regions upstream of the ATG start site of genes, blue slices are coding sequences/gene bodies of genes, and green slices are 3’ regions downstream of the stop codon of genes. (F) Zoom-in of the first 100kb region (left) and the last 100kb region (right) of chromosome two. Plotted are the ChIP-seq fold enrichment of PfMORC (top track; pink) and heterochromatin protein 1 (HP1; middle track; orange) with gene annotations (bottom track; blue bars; P. falciparum 3D7 strain, version 3, release 57; PlasmoDB.org).

(A) Mean fold enrichment (Log2[IP/Input]) of PfMORC, six associated factors (AP2-G5, AP2-O5, AP2-I, PF3D7_1107800, PF3D7_0613800, and PF3D7_1239200), HP1, and a negative no-epitope control across PfMORC binding sites at the 30 h timepoint. (B) Mean fold enrichment (Log2[IP/Input]) of PfMORC, six associated factors (AP2-G5, AP2-O5, AP2-I, PF3D7_1107800, PF3D7_0613800, and PF3D7_1239200), HP1, and a negative no-epitope control across PfMORC binding sites at the 40 h timepoint. (C) Mean fold enrichment (Log2[IP/Input]) and heatmap of two H3K36me2 epigenetic mark timepoints across PfMORC binding sites at 30 h. (D) Mean fold enrichment (Log2[IP/Input]) and heatmap of two H3K36me2 epigenetic mark timepoints across PfMORC binding sites at 40 h.

Transcriptome analysis of PfMORC knockdown revealed differential gene expression.

(A) Volcano plot displaying the differential gene expression in PfMORC-KD compared to the PfMORC-WT phenotype. Tightly synchronized PfMORCHA-glmS parasites (32 hpi ± 3 h) were split into two populations, one of which was treated with 2.5 mM GlcN to obtain the PfMORC knockdown phenotype and the other was not treated with GlcN to obtain wild-type phenotype. Total RNA-seq was performed, and significant threshold parameters for DEGs were assigned to a p-value < 0.05 and -log2 fold change > 1 from three biological replicates. (B) Scatter plot shows upregulated and downregulated DEGs which were further categorized for pathway and functional enrichment analysis using the KEGG database (p-adjusted value < 0.05). The circle size at the vertical axis represents the number of genes in the enriched pathways and the horizontal axis represents gene richness as a ratio of DEGs in the pathways to the total genes in a specific pathway. (C) The violin plot of log2 fold change of genes belonging to the multigene family is constructed from PfMORC-KD vs PfMORC-WT, which shows DEGs of multigene family proteins upon PfMORC knockdown. (D) The bar plot illustrates the upregulated DEGs of apical organelle origin in PfMORC-KD parasites involved in host cell invasion. (E) Venn diagram showing the comparison between genes obtained from ChIP-seq data and DEGs obtained from RNA-seq data. Both 30hpi and 40 hpi time points were taken for comparison and showed high overlap with each other but there was no overlap with RNA-seq data.

(A) Coomassie-stained 6% SDS‒PAGE gel showing the parasite lysate of wild-type 3D7 and PfMORCGFP after coimmunoprecipitation with anti-GFP magnetic beads. Both lanes were used for mass spectrometry analysis. (B) Histogram shows the total proteins identified in mass spectrometry analysis from three biological replicates in wild-type 3D7 and PfMORCGFP coimmunoprecipitated samples. Venn diagram illustrates the labeled free LC-MS/MS enrichment of peptide hits obtained from (C) 3D7 control and (D) from PfMORCGFP parasites lysate. Briefly, 32 hpi (±4 h) trophozoite stage parasites were harvested and lysed, followed by incubation with anti-GFP-Trap-A beads from three independent biological replicates were used for quantification. False discovery rate (FDR) of 1% and peptides ≥2 leads to identifying 191, 814, 589 and 211, 617, 656 significant proteins in 3D7 and PfMORCGFP, respectively. (E) MS/MS normalization of identified proteins from 3D7 parasites expressing PfMORC and transgenic parasites expressing GFP (PfMORCGFP) was carried out. Gene ontology classification showing biological process, cellular component, and molecular function of PfMORCGFP/3D7 normalized proteins showing fold change ≥ 1.5.

(A) Correlation plot (DeepTools PlotCorrelation) of the 30 h samples compared to the negative control ChIP-seq sample. (B) Correlation plot (DeepTools PlotCorrelation) of the 40 h samples compared to the negative control ChIP-seq sample. (C) Violin plot showing the ChIP-seq fold enrichment values of significantly called peaks in all 6 biological replicates. The two GFP samples were only used as additional controls for comparison purposes. (D) Venn diagram comparing the overlap of MACS2-called peaks between anti-HA biological replicates at 30 h. (E) Venn diagram comparing the overlap of MACS2-called peaks between anti-HA biological replicates at 40 h. (F) Venn diagram comparing the overlap of MACS2-called peaks between anti-GFP biological replicates at 40 h.

(Top) Profile plot of the mean PfMORC ChIP-seq fold enrichment (Log2[IP/Input]) for all four samples across all PfEMP1 (var) gene 5’ upstream regions and gene bodies. (Bottom) Heatmap of the PfMORC ChIP-seq fold enrichment (Log2[IP/Input]) for all four samples across all PfEMP1 (var) gene 5’ upstream regions and gene bodies. (Inset to the right) Zoom in on the average enrichment of PfMORC at var genes with annotated exons.

(Top) Profile plot of the mean PfMORC ChIP-seq fold enrichment (Log2[IP/Input]) for all four samples across all rif gene 5’ upstream regions and gene bodies. (Bottom) Heatmap of the PfMORC ChIP-seq fold enrichment (Log2[IP/Input]) for all four samples across all rif gene 5’ upstream regions and gene bodies. (Inset to the right) Zoom in on the average enrichment of PfMORC at rif genes with annotated exons.

(Top) Profile plot of the mean PfMORC ChIP-seq fold enrichment (Log2[IP/Input]) for all four samples across all rif gene 5’ upstream regions and gene bodies. (Bottom) Heatmap of the PfMORC ChIP-seq fold enrichment (Log2[IP/Input]) for all four samples across all rif gene 5’ upstream regions and gene bodies. (Inset to the right) Zoom in on the average enrichment of PfMORC at rif genes with annotated exons.

The heatmaps show the transcript abundance (Chappell et al. 2020) of putative PfMORC gene targets at 30 h (left) and 40 h (right). Red signifies high transcript abundance, and green signifies low transcript abundance. Both timepoints are organized into two major clusters (highlighted with the yellow bar and blue bar).

(A) Associated with Figure 3A,B. Mean fold enrichment (Log2[IP/Input]) summary plot (top) and full heatmap (bottom) of fold enrichment of PfMORC, six associated ApiAP2 factors (AP2-G5, AP2-O5, AP2-I, PF3D7_1107800, PF3D7_0613800, and PF3D7_1239200), HP1, and a negative no-epitope control across PfMORC binding sites at the 30 h and 40 h timepoints. (B) Quantitative Venn diagrams of the binding site overlaps between PfMORC and the six associated ApiAP2 factors.

DNA motif analyses from these different categories: (1) Unique to 30 hpi ChIP-seq Timepoint, (2) 30hpi ChIP-seq Timepoint, (3) Overlap of ChIP-seq Timepoints, (4) 40hpi ChIP-seq Timepoint, and (5) Unique to 30hpi ChIP-seq Timepoint. The values to the right of each motif contain the enrichment value, number of peaks containing that motif, and percent of the peaks the contain that motif calculated by Meme Suite.

Mean fold enrichment (Log2[IP/Input]) summary plot (top) and full heatmap (bottom) of fold enrichment of ten selected epigenetic marks (H2A.Z, H3K9ac, H3K4me3, H3K27ac, H3K18ac, H3K9me3, H3K36me2/3, H4K20me3, and H3K4me1) across PfMORC binding sites at the 30 h and 40 h timepoints.

Comparison of transcriptional changes with melatonin treatment.

(A) Volcano plot showing the differentially expressed genes in PfMORC-KD parasites relative to PfMORC-WT after 100 nM melatonin treatment for 5h from three independent experiments. (B) Venn diagram shows intersecting DEGs from the experiment with KD vs WT with DEGs obtained from the experiment (KD vs WT) treated with 100 nM melatonin for 5 h. Number of DEGs are shown as up (red) and downregulated (green). The intersecting region shows 282 upregulated and 340 downregulated genes. Heatmap showing significant DEGs based on p-values and log2FD for upregulating (C) and downregulating (D). These genes are taken from 622 intersecting DEGs showing partial changes in expression after melatonin treatment.