Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

SummaryHistone variants are important components of eukaryotic chromatin and can alter chromatin structure to confer specialized functions. H2B variant histones are rare in nature but have evolved independently in the phyla Apicomplexa and Trypanasomatida. Here, we investigate the apicomplexan-specific Plasmodium falciparum histone variant Pf H2B.Z and show that within nucleosomes Pf H2B.Z dimerizes with the H2A variant Pf H2A.Z and that Pf H2B.Z and Pf H2A.Z occupancy correlates in the subset of genes examined. These double-variant nucleosomes also carry common markers of euchromatin like H3K4me3 and histone acetylation. Pf H2B.Z levels are elevated in intergenic regions across the genome, except in the var multigene family, where Pf H2A.Z/Pf H2B.Z doublevariant nucleosomes are only enriched in the promoter of the single active var copy and this enrichment is developmentally regulated. Importantly, this pattern seems to be specific for var genes and does not apply to other heterochromatic gene families involved in red blood cell invasion which are also subject to clonal expression. Thus, Pf H2A.Z/Pf H2B.Z double-variant nucleosomes appear to have a highly specific function in the regulation of P. falciparum virulence.