Genome wide distribution of G-quadruplexes and their impact on gene expression in malaria parasites

Abstract

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite’s AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i. e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.

Author summary

Malaria persists as a global health concern and is caused by divergent eukaryotic parasites of the genus Plasmodium. These parasites have a complex life cycle during which stage transitions are characterised by tightly regulated cascades that ensure stage-specific gene expression, which are not yet fully understood. Here, we have performed a genome wide scan of G-quadruplex (G4) DNA motifs in the parasite’s AT-rich genome, using G4Hunter. We highlight a significant enrichment of G4s in nucleosome-depleted regions and in the first exon of var genes, which encode an important virulence factor associated with severe malaria. This is a pattern conserved among Plasmodium-related species of the Laverania subgenus. We then validate the regulatory function of G4s on transcription, using a luciferase reporter system. Finally, we show that the highly selective G4-stabilizing ligand pyridostatin causes large scale perturbations of the transcriptome in Plasmodium falciparum, thus showcasing their potential as antimalarials. Altogether, our study highlights G4s as a new layer of gene regulation in malaria parasites, and a likely potentiator of var gene diversity.