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

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 thevargene 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 thePlasmodium falciparumgenome 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 ofvargenes, a pattern that is conserved within the closely relatedLaverania Plasmodiumparasites. Under G4-stabilizing conditions,i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that abona fideG4 found in the non-coding strand ofvarpromoters 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 inPlasmodiumgene expression regulation, sub-telomeric recombination andvargene biology. Author summary Malaria persists as a global health concern and is caused by divergent eukaryotic parasites of the genusPlasmodium. 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 ofvargenes, which encode an important virulence factor associated with severe malaria. This is a pattern conserved amongPlasmodium-related species of theLaveraniasubgenus. 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 inPlasmodium 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 ofvargene diversity.