Functional analysis of epigenetic regulation of tandem RhopH1/clag genes reveals a role in Plasmodium falciparum growth.

The Plasmodium RhopH complex is a high molecular weight antigenic complex consisting of three subunits - RhopH1/clag, RhopH2 and RhopH3 - located in the rhoptry secretory organelles of the invasive merozoite. In Plasmodium falciparum RhopH1/clag is encoded by one of five clag genes. Two highly similar paralogous genes, clag 3.1 and clag 3.2, are mutually exclusively expressed. Here we show clonal switching from the clag 3.2 to the clag 3.1 paralogue in vitro. Chromatin immunoprecitation studies suggest that silencing of either clag 3 paralogue is associated with the enrichment of specific histone modifications associated with heterochromatin. We were able to disrupt the clag 3.2 gene, with a drug cassette inserted into the clag 3.2 locus being readily silenced in a position-dependent and sequence-independent manner. Activation of this drug cassette by drug selection results in parasites with the clag 3.1 locus silenced and lack full-length clag 3.1 or 3.2 transcripts. These clag 3-null parasites demonstrate a significant growth inhibition compared with wild-type parasites, providing the first genetic evidence for a role for these proteins in efficient parasite proliferation. Epigenetic regulation of these chromosomally proximal members of a multigene family provides a mechanism for both immune evasion and functional diversification.