Investigating the mechanism of Heterochromatin Protein-1 (HP1) mediated var gene regulation in Plasmodium falciparum

Abstract

Malaria caused by the Plasmodium falciparum is driven by alterations in the infected RBC surface by recruiting antigens that can bind to the endothelial receptors (cytoadherence). The clonally variant multigene family of genes known as var genes (~60 members) codes for proteins that cause cytoadherence. The var gene family shows mutually exclusive expression even though the mechanism of regulation is unclear. Interestingly, var genes are marked by a unique set of activation (histone acetylations) and repression (H3K9me3) marks, some of which are exclusive for virulence family genes. PfHP1 (P. falciparum Heterochromatin Protein 1), a homolog of HP1, specifically binds to H3K9me3 histone modification. ChIP sequencing of PfHP1 has revealed a restricted pattern of heterochromatin spread across all the var introns that is correlated with the intronic transcription from there. These results indicate that PfHP1 shows a reversible spread across the var genes that may enable activation of some var genes while others are kept suppressed. But the mechanism of PfHP1 dependent spread of heterochromatin is not very well known. In HP1 homologs of higher eukaryotes it is shown that heterochromatinization is facilitated by liquid-liquid phase separation (LLPS). We further explored the property of phase separation of PfHP1 in vitro to understand the biochemical basis of heterochromatinization. Our data suggest that the PfHP1 phase separates in vitro in an RNA-dependent manner. Higher concentrations of AU-rich non-coding RNA (notably var introns are ~ 80 % AT-rich) causes the dissolution of PfHP1 droplets. Our single molecular DNA tethering experiments showed that PfHP1 preferably forms puncta over AT-rich intronic DNA and compacts the DNA. We have also identified point mutations in PfHP1 that disrupts the phase separation in vitro. We have generated P. falciparum transgenics with these mutations to further study the effect of PfHP1 phase separation in vivo and on var gene expression. Overexpression of PfHP1 mutants showed reduced chromatin binding on var genes. Conditional expression of PfHP1 point mutations showed dispersed nuclear localization as opposed to puncta-like appearance of the wild type protein. In addition some of these point mutations showed growth defects and gametocytogenesis in the parasites. These observations indicate a strong possibility that disruption of phase separating properties of PfHP1 in vivo might affect its gene regulatory function. Our study reveals the nature of PfHP1 mediated heterochromatin formation and its role in var gene regulation.