PfMORC-HA is associated with heterochromatin.

(a) Illustration of the PfMORC containing domains including Kelch type b propeller and ATPase domains using InterProScan. (b) Design strategy applied for PfMORC C-terminal HA tagging. PCR amplification of the genomic C-terminus end of Pfmorc region extending towards the 3’UTR (F1) as well as extension from the C-terminus towards the HA flanking sequence (F2) verifies the correct insertion site. NF54 genomic DNA was used as negative control. (c) IFA experiment: PfMORC foci 22 expressing co-localization with H3K9me3 marks (red). Cell nuclei are stained with DAPI (blue). BF: brightfield (d) Protein immunoprecipitation: Significance plot representing PfMORC interactome recovered through immunoprecipitation followed by mass spectrometry (IP-MS). Graph list MORC (orange) bindings partners of highest affinity associated with TF regulation 22 22 and chromatin remodelers, erasers, and writers (purple). Proteins enriched in the PfMORC-HA samples compared with controls were filtered with log2 FC ≥ 2 and Z statistic > 5. (e) Venn diagram representing overlapping proteins identified among five publications. Values represent the total number of significant proteins identified as overlapping between two subsets. (f) Gene ontology enrichment analysis of the significantly enriched proteins. The top 2 terms of Cellular Component (top) and top 3 terms of Molecular Function (bottom) are represented as -Log10 (adjusted P-value) (Fisher’s exact test with Bonferroni adjustment).

Genome wide distribution of PfMORC proteins.

(a) Chromosome distribution plots of PfMORC binding showing a predisposition for subtelomeric and internal var gene regions (red). Each track is input subtracted, and per-million read count normalized before normalizing the track height to allow for direct comparison between stages. Grey boxes indicate the position of centromeres. (b) Overlap of called peaks between time points. (c) Profile plots showing PfMORC coverage from 0.5 kb 5’ of the transcription start site (TSS) to 1.5 kb 3’ of the TSS in the ring, trophozoite, and schizont stages. Each plot includes per-million read count normalized coverage at 1 bp resolution for all genes within the var and rifin gene families as well as gametocyte-specific genes. (d) PfMORC coverage of the gametocyte-specific transcription factor ap2-g.

PfMORC is essential for cell survival.

(a) Diagram representation of PfMORC-HA-TetR-DOZI plasmid. (b). PCR amplification is used to verify genomic insertion using primers sets targeting 1.5 kbp of WT Pfmorc genome locus absent in transgenic line (MORC construct) (F3) as well as verification of HA insertion (F4) and TetR-DOZI system extending along 3’ UTR of the construct (F5). (c) Phenotypic and (d) quantitative analysis of parasite cell progression after aTC withdrawal at the ring stage (0-6 hpi) (2-way ANOVA, n=3, p≤0.0001). (e) Phenotypic and quantitative (f) analysis of parasite cell progression after aTC removal at the trophozoite stage of cell cycle progression (24 hpi) (2-way ANOVA, n=3, p≤0.0001).

PfMORC KD on parasite transcriptome.

Volcano plots denoting upregulated (red), and downregulated (blue) genes discovered through differential expression analysis following PfMORC knockdown at (a) 24 hpi (b) 36 hpi. Gene ontology enrichment analysis for upregulated (red) and downregulated (blue) genes at (c) 24 hpi and (d) 36 hpi. (e) Overlap of differentially expressed genes and genes containing significant peaks called by PfMORC ChIP-seq analysis at the trophozoite and schizont stages.

Impact of PfMORC KD on heterochromatin markers.

(a) Genome-wide H3K9me3 coverage of IGG subtracted and per-million normalized (+/-) aTC show similar distribution and concentration within telomeres and antigenic gene clusters highlighted in red. Replicates (n = 2) are merged using the mean of the normalized read coverage per base pair. (b) Binned coverage of var and rifin genes from 1 kb upstream of the TSS to 1 kb downstream of the end. The exons and intron of all genes within these families are split into five equal sized bins and the 5’ and 3’ regions are binned into five 200 bp bins. Read counts within each bin are per-million and bin length normalized prior to plotting. (c) Coverage of the five most upregulated var and rifin genes as determined by the transcriptomic analysis which shows elevated coverage in (+) aTC cells.

Loss of PfMORC expression correlates with heterochromatin expansion.

Intrachromosomal interaction heatmaps of (+/-) aTC PfMORC for chromosome 4 at (a) 24 hpi and (b) 36 hpi displaying heterochromatin clustering within antigenic (var, rifin and stevor) gene-dense regions (red). Differential interaction heatmaps highlight changes in chromatin structure following removal of aTC and subsequent PfMORC knockdown at (c) 24 hpi and (d) 36 hpi. (e) Whole-genome 3D models of the chromatin structure at both time points (24 hpi and 36 hpi) and (+/-) aTC.