Genome-wide profiling of histone modifications in Plasmodium falciparum using CUT&RUN

Abstract

We recently adapted a CUT&RUN protocol for genome-wide profiling of chromatin modifications in the human malaria parasite Plasmodium Using the step-by-step protocol described below, we were able to generate high-quality profiles of multiple histone modifications using only a small fraction of the cells required for ChIP-seq. Using antibodies against two commonly profiled histone modifications, H3K4me3 and H3K9me3, we show here that CUT&RUN profiling is highly reproducible and closely recapitulates previously published ChIP-seq-based abundance profiles of histone marks. Finally, we show that CUT&RUN requires substantially lower sequencing coverage for accurate profiling compared with ChIP-seq.

Figure 1.. Overview of genome-wide profiling of histone modifications using CUT&RUN.

(A) Infected RBCs are immobilized on concanavalin-coated beads. (B) Immobilized cells are permeabilized, allowing histone modification-specific antibody to diffuse into the nucleus to bind to its targets. (C) After removal of excess antibodies, MNase fused to protein A/G is added under low calcium conditions and binds to chromatin-associated antibodies. (D) After removal of excess proteinAG–MNase, cleavage adjacent to bound nucleosomes is activated by addition of calcium-cations. (E, F) Liberated nucleosomes are allowed to diffuse out of the permeabilized cells for subsequent (F) DNA extraction, library generation, high-throughput sequencing, and analysis.

Figure 2.. CUT&RUN profiles of histone modifications are highly reproducible.

Fold Enrichment profiles of H3K9me3 (green) and H3K4me3 (purple) in three biological replicates. H3K4me3 and H3K9me3 tracks were scaled to maximal value in the region shown. Example locus on chromosome 8 contains a non-subtelomeric heterochromatin island. Genes shown in blue are encoded on the top (+) strand, whereas those in red are encoded on the bottom (−) strand. Gene IDs are shown to the right of the gene.

Figure S1.. Correlation of fold enrichment between CUT&RUN replicates and published ChIP-seq data.

Color and values shown indicate the genome-wide Pearson’s correlation coefficient.

Figure 3.. Comparison of H3K4me3 and H3K9me3 profiles obtained by CUT&RUN to published ChIP-seq profiles at an example locus containing a non-subtelomeric heterochromatin island on chromosome 8.

Tracks show the relative fold enrichment of specific histone modifications, H3K9me3 and H3K4me3, and for heterochromatin protein 1, the H3K9me3 histone reader, versus either isotype controls for CUT&RUN or input DNA (ChIP-seq). Sequence reads from published stage-matched H3K9me3 (SRR4444647, SRR4444639), HP1 (SRR5935737, SRR5935738), and H3K4me3 (SRR065659, SRR065664) ChIP-seq experiments were downloaded from the NCBI Sequence Read Archive. Genes shown in blue are encoded on the top (+) strand, whereas those in red are encoded on the bottom (−) strand. Gene IDs are shown to the right of each gene.

Figure S2.. Comparison of H3K4me3 and H3K9me3 profiles obtained by CUT&RUN to published ChIP-seq profiles for full length chromosomes 4 and 7.

Tracks show the relative fold enrichment of specific histone modifications, H3K9me3 and H3K4me3, and for heterochromatin protein 1, the H3K9me3 histone reader, versus either isotype controls for CUT&RUN or input DNA (ChIP-seq). Sequence reads from published stage-matched H3K9me3 (SRR4444647, SRR4444639), HP1 (SRR5935737, SRR5935738), and H3K4me3 (SRR065659, SRR065664) ChIP-seq experiments were downloaded from the NCBI Sequence Read Archive.

Figure 4.. Fold Enrichment profiles of down-sampled H3K9me3 (green) and H3K4me3 (purple) CUT&RUN reads show good signal-to-noise ratios down to ∼1 million read-pairs per sample.

Read pairs were serially down-sampled in silico by fourfold to evaluate the signal-to-noise ratio. Each track is scaled to its maximal value across the locus shown. The number of sequenced paired-end 50 bp (PE50) clusters used is indicated in millions. Example locus on chromosome 8 contains a non-subtelomeric heterochromatin island. Genes shown in blue are encoded on the top (+) strand, whereas those in red are encoded on the bottom (−) strand. Gene IDs are shown the above or below the gene. Grey regions in the 64× down-sampled tracks lack coverage and are inferred based on neighboring bins.

Figure 5.. iRBCs treated with 0.025% digitonin and stained with Hoechst 33342 and propidium iodide.

Nuclei of permeabilized cells will stain with both dyes. Scale bar, 4 µm.

Figure 6.. Quantification of parasite genome content.

Left: flow cytometric analysis of live cultures after staining for 30 min with Hoechst 33342 and thiazole orange allows easy differentiation of uninfected RBCs (red), ring-stage infected cells (blue), and late-stage infected cells (orange) based on RNA and DNA content. Right: 1N ring stages can be used as the reference for calculating genome content of late stages.

Figure 7.. Example size distributions of CUT&RUN libraries from H3K9me3, H3K4me3, IgG isotype, and PCR negative control as analyzed by TapeStation.

Primer dimer, adapter, and library peaks are indicated in the top left panel.