Genome-wide mapping of DNA G-quadruplexes in Trypanosoma brucei chromatin reveals enrichment in coding regions

G-quadruplexes (G4s) are non-canonical DNA structures formed in guanine-rich sequences that are proposed to act as regulatory elements in trypanosomatid parasites, including Trypanosoma brucei , the causative agent of African sleeping sickness. However, their functional roles remain poorly understood, largely due to limited knowledge of their genomic distribution. Herein, we performed computational analyses across 63 trypanosomatid species uncovering high degree of variability in G4-prevalence and species-specific patterns. We generated the first genome-wide map of G4s in T. brucei using G4 chromatin immunoprecipitation followed by sequencing (G4 ChIP-Seq), which revealed a striking enrichment of G4s within coding DNA sequences (CDSs). This pattern diverges markedly from in silico predictions and previous genome-wide G4 mapping studies in humans, suggesting that G4s may play unique roles exclusive to trypanosome biology. To investigate their functional relevance, we profiled the transcriptome of T. brucei upon treatment with the G4-stabilising ligand PhenDC3. We observed that PhenDC3 exerts targeted gene expression perturbation of genes bearing G4s, particularly those located within coding CDSs, where G4s are mostly enriched. Altogether, our findings highlight a distinctive role for G4s in the regulation of gene expression in T. brucei and support their potential as therapeutic targets in the treatment of African sleeping sickness.