Generation of parasites with a homogenous placental binding phenotype and consequential dominant expression of var2csa. (A) The presence of in vivo relevant knob-structures on pRBCs was confirmed by SEM prior to phenotypic selection. Shown is a representative image of a pRBC with knobs appearing as white protrusions. (B) Flow cytometry-based antibody recognition of VAR2CSA surface expression on repeatedly selected pRBCs. NF54 ptefKO served as negative control, NF54 was the original and unselected parasite line, NF54CSAm was an intermediately selected line and NF54CSAh was panned enough to achieve a homogenous population of VAR2CSA expressing pRBCs. (C) Representative image of a Giemsa stained placental section with bound NF54CSAh pRBCs. Tissue boundaries are indicated by white dashed lines. (D) Quantification of relative placental binding of NF54CSAh from three biological replicates. pRBCs bound per mm2 were normalized to the VAR2CSA negative NF54 ptefKO parasite line (n=3). (E) Spearman correlation of var gene expression profiles determined by RNA-seq for three bio-replicates each of NF54CSAh and the original, unselected NF54. (F) Differential gene expression between NF54CSAh and NF54 identified a limited number of significant genes (FDR <0.05, Log2 fold change >1) of which the majority were var genes (var2csa in blue, all other var genes in green). (G) Normalized read counts for all var genes revealed NF54 to be phenotypically highly heterogeneous whereas var2csa was the only var gene expressed by NF54CSAh.

The var2csa locus is devoid of repressive H3K9me3 in placental binding parasites. (A) Chromosomal distribution of repressive H3K9me3 as determined by ChIP-seq from merged biological triplicates of NF54 and NF54CSAh. Regional clusters of antigenically variable genes are noted with green below each plot. The only noticeable difference between the parasite lines was located to the left arm of chromosome 12, which contains the var2csa locus (dashed box). (B) Heatmaps of H3K9me3 occupancy in NF54 and NF54CSAh for all var genes (0.5 kb upstream of the transcription start site (TSS) and 1.5kb into the gene body) with var2csa displayed with bolded gene ID. (C and D) H3K9me3 enrichment in the antigenically variable multigene families var, rif, stevor and pfmc-2tm partitioned into 5’ UTR, exons and introns versus the rest of the genomes (all other genes and intergenic regions) of NF54 and NF54CSAh.

Changes in chromatin organization and perinuclear repositioning upon var2csa activation and silencing. (A-C) Hi-C generated and normalized contact counts representing intrachromosomal interactions for chromosome 2, 4 and 12 in NF54 (left panels) and NF54CSAh (middle panels). Differential intrachromosomal interactions between the two parasite lines (right panels) displayed a general increase in interactions between subtelomeric and chromosome internal var gene clusters for NF54CSAh compared to NF54. Chromosome 12 (C) stood out as the only exception with decreased interactions in NF54CSAh for the subtelomere containing the var2csa locus. (D) 3D chromatin modeling for NF54 displays a polarized nucleus with clustering of centromeres (blue) and telomeres (red) in distinct regions. The majority of var genes (green), including var2csa (blue), are located in close vicinity to the telomeric cluster. (E) The 3D chromatin model for NF54CSAh displayed a similar topology to NF54 with the exception of decreased distance between var genes (green) and telomere ends (red) over-all and an increased distance of var2csa (blue) from the telomeric cluster.

Regional differences in 5mC occupancy tracks with global transcriptional activity but is dissociated from var2csa regulation. (A) Genome-wide distribution of input normalized MeDIP-seq counts for 5mC in NF54 and NF54CSAh reveal a high level of concordance between the analyzed parasite lines. Regional clusters of antigenically variable genes are denoted in green below each plot with an area of chromosome 12 marked by a dashed box and a zoomed-in insert displaying minor differences in 5mC occupancy in the var2csa locus between NF54 and NF54CSAh (var2csa in blue, other var genes in green and rif in black). (B) Levels of 5mC marks binned in 5’ flanks and first exon for the top 20% highest expressed genes and 20% lowest expressed genes in NF54 and NF54CSAh. (C) Distribution of 5mC across exon-intron boundaries for the 5mC enriched var, rif and other two-exonic genes.