Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

Abstract

Post-translational modifications of histone H3 N-terminal tails are key epigenetic regulators of virulence gene expression and sexual commitment in the human malaria parasite Plasmodium falciparum Here, we identify proteolytic clipping of the N-terminal tail of nucleosome-associated histone H3 at amino acid position 21 as a new chromatin modification. A cathepsin C-like proteolytic clipping activity is observed in nuclear parasite extracts. Notably, an ectopically expressed version of clipped histone H3, PfH3p-HA, is targeted to the nucleus and integrates into mononucleosomes. Furthermore, chromatin immunoprecipitation and next-generation sequencing analysis identified PfH3p-HA as being highly enriched in the upstream region of six genes that play a key role in DNA replication and repair: In these genes, PfH3p-HA demarcates a specific 1.5 kb chromatin island adjacent to the open reading frame. Our results indicate that, in P. falciparum, the process of histone clipping may precede chromatin integration hinting at preferential targeting of pre-assembled PfH3p-containing nucleosomes to specific genomic regions. The discovery of a protease-directed mode of chromatin organization in P. falciparum opens up new avenues to develop new anti-malarials.

Keywords: epigenetics; histone H3 clipping; malaria.

Figure 1. Plasmodium falciparum nucleosomal histone H3 is proteolytically processed in a stage‐specific manner between amino acids 21 and 22

1. Immunoblot analysis of nuclear and cytoplasmic extracts prepared from parasites synchronized at ring (R), trophozoite (T), and schizont (S) stages with anti‐histone H3 C‐terminus antibodies identifies three bands: full‐length histone H3, an intermediate form (H3‐int), and a fully processed form (H3p). Anti‐histone H4 and anti‐PfAldolase antibodies served as loading controls for the nuclear and cytoplasmic fraction, respectively.

2. Immunoblot assays of mononucleosomes with anti‐histone H3 C‐terminus or anti‐histone H4 antibodies. The right panel shows a mononucleosome preparation separated on a denaturing polyacrylamide gel and stained with Coomassie Brilliant Blue (C.B.).

3. Immunoblot analysis of a schizont‐stage histone preparation with antibodies against the indicated post‐translational modification (PTM) in the N‐terminal tail of histone H3 (upper panel).

4. Results from the Edman degradation analysis of full‐length PfH3 or processed PfH3p; the arrow indicates the position of histone H3 clipping.

Source data are available online for this figure.

Figure 2. A cathepsin C‐type protease in schizont‐stage nuclear extracts may mediate Plasmodium falciparum histone H3 processing

1. Schematic representation of the in vitro proteolysis assay developed in this study. N‐terminally GST‐tagged PfH3 was incubated with schizont‐stage nuclear extracts at 37°C for 3 h and analyzed by immunoblotting with anti‐GST antibodies. GST‐tagged PfH2A served as a control.

2. GST‐PfH3 or GST‐PfH2A was incubated with increasing amounts of nuclear extracts, and the reactions were resolved using denaturing gel electrophoresis and analyzed by immunoblotting with anti‐GST antibodies.

3. GST‐PfH3 was incubated with 25 μg of nuclear extracts and the indicated protease inhibitor. The resulting products were analyzed by denaturing gel electrophoresis and immunoblotting with anti‐GST antibodies. (−) indicates a reaction without nuclear extract and protease inhibitor.

4. Target profile of the protease inhibitors used in panel (C).

Source data are available online for this figure.

Figure 3. Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes

1. Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm.

2. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies.

3. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies.

Source data are available online for this figure.

Figure 4. In Plasmodium falciparum, clipped histone H3 is targeted to the 5′UTR of genes regulating DNA replication

1. Genome‐wide distribution of ectopically expressed PfH3p‐HA in P. falciparum schizont stages is represented as fold enrichment of PfH3p‐HA ChIP‐seq signal over input (PfH3p‐HA/input in blue; y‐axis scale 1–10) or over PfH3n (PfH3p‐HA/input in teal; y‐axis scale 1–10) calculated using MACS2. The coverage of PfH3p‐HA (red; y‐axis scale 0–50), input (black; y‐axis scale 0–30), and PfH3n (green; y‐axis scale 0–30) is also shown and represents average reads per million over 1,000 nt bins of the genome. The x‐axis represents chromosome size in Mb. Major MACS2‐derived peaks identified in all replicates are indicated using an orange arrowhead.

2. Principal component analysis of the different ChIP‐seq replicates (bigwig files derived from deduplicated bam files) was performed using the plotPCA function of deepTools on a multibigwig summary file, over 150 nt bins. The Eigenvalues of the top two principal components PC1 and PC2 are shown, and meaningful clustering of replicates is highlighted. Rep = biological replicate.

3. Summary of the PfH3p‐enriched peaks—and corresponding genes—identified in the PfH3p‐HA versus input or PfH3p‐HA versus PfH3n comparisons using MACS2 peak‐calling analysis. The overlap between the two comparisons is indicated using a Venn diagram. FE = fold enrichment; q‐value represents the Benjamini and Hochberg false discovery rate.

4. The genomic context of the six peaks identified in all three PfH3p‐HA ChIP‐seq replicates is shown. The fold enrichment of PfH3p‐HA ChIP‐seq signal over input (PfH3p‐HA/input; blue) or PfH3n (PfH3p‐HA/PfH3n; teal) is shown, as are the coverage plots of PfH3p‐HA (red), PfH3n (green), and input (black), which are represented as average reads per million over 1,000 nt bins of the genome. DNA replication genes: replication protein A 1 (RPA1), proliferating cell nuclear antigen 1 (PCNA1), single‐stranded DNA‐binding protein (SSB), topoisomerase I (TOPO‐I), DNA polymerase alpha subunit (DNA pol a), and topoisomerase II (TOPO‐II) are highlighted and their directionality indicated. Note that the data in part D correspond to Biological Replicate 1.