Figures and data
– Proteomic analysis of parasites expressing PfMORCGFP reveals PfMORC association with nuclear proteins of epigenetic regulation.
(A) Venn diagram depicting protein enrichment from three biological replicates using PfMORC as bait from 32 hpi (±4 h) trophozoite stage PfMORCGFP parasite lysate with anti-GFP-Trap-A beads. (B) Venn diagram was created for CoIPed protein in PfMORCGFP with selected proteins from recently published work by Hillier et al, Bryant et al. and Subudhi et al. Intersecting proteins showing the majority of proteins are AP2 and chromatin remodellers. (C) Protein-protein interaction network of enriched proteins co-IPed with anti-GFP and detected by mass spectrometry was generated with known PfMORC interactome from published work. The String database and Cytoscape were used to create an interactive protein-protein interaction network.
– 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 showing the differential gene expression in PfMORC-KD compared to the PfMORC-WT phenotype. Tightly synchronized parasites were treated with 2.5 mM GlcN to obtain the PfMORC knockdown phenotype and another without GlcN treatment. Total RNA was collected from three biological replicates, and RNA-seq was performed. Significant threshold parameters for DEGs were assigned to a p value < 0.05. (B) Up and downregulated DEGs were further functionally categorized using KEGG database. (C) The violin plot of log2FoldChange of genes belonging to the multigene family is constructed from PfMORC-KD vs PfMORC-WT shows DEGs of multigene family proteins upon PfMORC knockdown. (D) The bar plot shows upregulated DEGs of apical organelle origin in PfMORC-KD parasites that are 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.
