Phase separation of heterochromatin protein 1 regulates virulence gene expression in Plasmodium falciparum

Plasmodium falciparum malaria relies on antigenic variation mediated by mutually exclusive expression of var genes. Repressed var genes cluster together and are bound by PfHP1 (Plasmodium falciparum heterochromatin protein 1), but the regulatory mechanism remains unclear. Here, we show that PfHP1 undergoes phase separation in vitro and compartmentalizes DNA. This process is tunable by RNA and PfH2A.Z. Our single-molecule assays show that PfHP1 preferentially compacts AT-rich DNA. We identify point mutations in the intrinsically disordered region of PfHP1 that disrupt phase separation and DNA compaction. Analysis of GFP-PfHP1 parasites by fluorescence recovery after photobleaching shows rapid recovery, consistent with fluid condensate behavior in vivo. Expression of phase-separation-defective mutants cause dispersed nuclear localization, altered chromatin binding, and derepression of up to 54 of 60 var genes. Hi-C analysis reveals loss of heterochromatic interactions, establishing PfHP1-mediated phase separation as a key mechanism for heterochromatin organization and var gene silencing.