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.

Author Summary

Plasmodium falciparum causes the most severe form of malaria in humans. The high virulence of this unicellular parasite is in part related to the selective expression of members of falciparum-specific gene families. These genes encode proteins that are exported into the cytoplasm and onto the surface of infected red blood cells. To avoid recognition by the host's immune system, P. falciparum employs sequential expression of antigenically different variants of these surface proteins. While the epigenetic mechanisms responsible for such clonal expression have been studied in some detail for the major virulence gene family var, the regulation and function of other exported protein families remain elusive. Here, we identify P. falciparum heterochromatin protein 1 as a major structural component of virulence gene islands throughout the parasite genome. This factor binds specifically to a reversible histone modification, which marks these virulence loci for transcriptional silencing. Our observations suggest a unifying epigenetic strategy in the regulation of host–parasite interactions and immune evasion in P. falciparum. Furthermore, these findings have important implications for the future study of hitherto uncharacterized exported proteins with roles in parasite virulence.