Abstract
SummaryHeterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium.
Highlights
Guided by a phylogenetic tree constructed from heterochromatin protein 1 (HP1) orthologs (Figure S1), we generated additional polyclonal rabbit antibodies against PvHP1 and PbHP1
Immunofluorescence assays (IFAs) using these antibodies visualized punctate signals in the nuclei of all species that are reminiscent of the perinuclear chromosome end clusters observed in P. falciparum (Brancucci et al, 2014) (Figures 1A and S1)
In western blot analyses these antibodies detected a protein of the expected size of HP1 in each species (Figure S1)
Summary
METHOD DETAILSP. falciparum Sample Collection and Chromatin Preparation Chromatin from 3D7 ring stages (8-16 hpi), trophozoites (32-30 hpi) or schizonts (40-48 hpi) (approximately 0.75-1.5x109 parasites each) and from Pf2004/T164dTom schizonts, stage II/III gametocytes (day four after re-invasion) or stage IV/V gametocytes (day nine after re-invasion) (approximately 2x108 parasites each) was prepared by crosslinking cultures with 1% formaldehyde (Sigma-Aldrich) for 15 min at 37C. Nuclei were isolated by releasing parasites from iRBCs using 0.05% saponin followed by lysis in CLB (20 mM Hepes, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.65% NP-40, 1mM DTT, 1x protease inhibitor (Sigma-Adrich), pH 7.9). Chromatin from NF54 and NF135 schizont stages (approximately 2-4x109 parasites each) was prepared by passing the cultures through Plasmodipure filters (EuroProxima) to remove white blood cells prior to formaldehyde crosslinking for 10 min at 37C and quenching in 0.125 M glycine. Nuclei were isolated by releasing parasites from iRBCs using 0.05% saponin followed by gentle homogenisation (pestle B, 15 strokes) in CLB2 (10 mM Tris-HCl, 3 mM MgCl2, 0.2% NP40, 1x protease inhibitor (Sigma-Adrich), pH 8.0) and centrifugation through a 0.25 M sucrose cushion (in CLB2) at 2000 rpm for 10 min at 4C. Chromatin fragment sizes ranged from 100-600 bp as determined by de-crosslinking a 50 ml aliquot and running the purified DNA on a 1% agarose gel
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