Plasmodium falciparum Heterochromatin Protein 1 Marks Genomic Loci Linked to Phenotypic Variation of Exported Virulence Factors

Abstract

Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that P. falciparum heterochromatin protein 1 (PfHP1) binds specifically to H3K9me3 but not to other repressive histone methyl marks. Based on nuclear fractionation and detailed immuno-localization assays, PfHP1 constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with virulence gene arrays in subtelomeric and chromosome-internal islands and a high correlation with previously mapped H3K9me3 marks. These include not only var genes, but also the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host–parasite interactions. In addition, we identified a number of PfHP1-bound genes that were not enriched in H3K9me3, many of which code for proteins expressed during invasion or at different life cycle stages. Interestingly, PfHP1 is absent from centromeric regions, implying important differences in centromere biology between P. falciparum and its human host. Over-expression of PfHP1 results in an enhancement of variegated expression and highlights the presence of well-defined heterochromatic boundaries. In summary, we identify PfHP1 as a major effector of virulence gene silencing and phenotypic variation. Our results are instrumental for our understanding of this widely used survival strategy in unicellular pathogens.

Transcriptional variation in the malaria parasitePlasmodium falciparum

“…Many of the genes that showed variant expression were found to be positive for the heterochromatin marks H3K9me3 and HP1 in previous chip on ChIP-based mapping studies (Supplemental Table S2; Flueck et al 2009;Lopez-Rubio et al 2009;Salcedo-Amaya et al 2009). Only two of the 28 variant gene families included in Table 1 were HP1-and H3K9me3-negative.…”

“…Likewise, of 35 gene families reported to be marked by HP1 or H3K9me3, only five small families did not have any member showing clonally variant ex- Table S2). c Gene families with >30% (+) or some, but <30% (+/À) of the members marked by HP1 or H3K9me3 (Flueck et al 2009;Lopez-Rubio et al 2009;Salcedo-Amaya et al 2009). d Gene families with >50% of the members located at subtelomeric regions (<150 kb from the chromosome end) are indicated as positive.…”

“…Gene families are detailed in Supplemental Table S4. The tracks at the right indicate whether a gene was included in the list of variant genes (Supplemental Table S2), whether it was positive for heterochromatin marks HP1 or H3K9me3 in any of the published reports (Flueck et al 2009;Lopez-Rubio et al 2009;Salcedo-Amaya et al 2009), and whether it is subtelomerically located (<150 kb from a chromosome end). Genes that show high aMAFC in some of the tracks but are not in the list of variant genes were excluded because changes in expression were attributable to CNVs, or for other reasons detailed in the text.…”

PfSec13 is an unusual chromatin associated nucleoporin of Plasmodium falciparum, which is essential for parasite proliferation in human erythrocytes

“…Within this unusual epigenome heterochromatin is designated to be enriched with specific heterochromatin marks such as H3K9me3 and PfHP1 (Chookajorn et al, 2007;Lopez-Rubio et al, 2009;Salcedo-Amaya et al, 2009). H3K9me3 and PfHP1 were shown to be associated with each other at distinct foci at the nuclear periphery (Flueck et al, 2009;Pérez-Toledo et al, 2009). In addition, these heterochromatin marks were implicated in silencing and epigenetic memory of virulence genes located at the nuclear periphery (Flueck et al, 2010).…”

“…Scale bar: 2 mm. (B) In vivo imaging of parasites expressing both PfSec13-GFP and PfHP1-HA-mCherry (Flueck et al, 2009) during the IDC. Although PfSec13 (green) and PfHP1 (red) localized to the nuclear periphery they do not co-localize.…”