Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite (2020)
Figure 4

Figure 4. ISWI is a transcriptional activator involved in var gene regulation.

Western blot analysis of cytoplasmic (Cty) and nuclear extracts from a bulk population of ISWI-3HA-ribo parasites in the absence (-) or presence (+) of glucosamine (GlcN). ISWI-3HA is detected with an anti-HA antibody. Antibodies against aldolase (Ald.) and histone H3 are controls for the cytoplasmic and nuclear extracts, respectively. Molecular weights are shown to the right.

MA plot of log2[glucosamine-treated/untreated, M] plotted over the mean abundance of each gene (A) at 12 hpi. Transcripts with a significantly higher (above x-axis) or lower (below x-axis) abundance in the presence of glucosamine are highlighted in red (q ≤ 0.05). iswi is highlighted in blue (q = 6.71x10-10) and the active var gene is highlighted in green (q = 2.53x10-5). Two and three replicates were used for untreated and glucosamine-treated parasites, respectively. p-values were calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected p-value.

Frequency plot showing the time in the IDC of peak transcript level [comparison to microarray time course in (Bozdech et al, 2003; Data ref: Bozdech et al, 2003)] for genes that are significantly down-regulated (blue) or up-regulated (yellow) following ISWI knockdown.

Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for genes that are down- (blue) or up-regulated (yellow) upon ISWI knockdown. One replicate was used for the ISWI ChIP-seq.

ChIP-seq data show enrichment of ISWI (blue) in clonal ISWI-3HA parasites at 12 hpi relative to regions of accessible chromatin ("ATAC," red). Chromatin accessibility (ATAC-seq) data were taken from the 15 hpi time point in (Toenhake et al, 2018; Data ref: Toenhake et al, 2018). Genome location is indicated at the top of the panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is enrichment (ChIP/Input or ATAC-seq/genomic DNA). One replicate was used for the ISWI ChIP-seq.

Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for all genes, which are grouped into quartiles based on their transcript levels (RPKM) at 12 hpi. Dark red represents genes with the highest transcript levels ("1st"), light red represents genes with the second highest transcript levels ("2nd"), orange represents genes with the third highest transcript levels ("3rd"), and yellow represents genes with the lowest transcript levels ("4th"). One replicate was used for the ISWI ChIP-seq and the transcription data are an average from the two replicates of the untreated ISWI-3HA clone used for the differential expression analysis.

ChIP-seq data show enrichment of ISWI (blue) and H3K9ac (green) in clonal ISWI-3HA parasites at 12 hpi at the active var gene (PF3D7_1240600). RNA-seq data from this clone at 12 hpi show transcript levels for this gene (grey). Genome location is indicated at the top of the panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is ChIP/Input for ChIP data and RPKM for RNA-seq data. One replicate was used for each ChIP-seq and the RNA-seq data is from a single replicate from the untreated ISWI-3HA clone used for the differential expression analysis.

Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for the active var gene (PF3D7_1240600, blue) or silent var genes (yellow). One replicate was used for the ISWI ChIP-seq.

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