Genome-wide mapping of DNA G-quadruplexes in Trypanosoma brucei chromatin reveals enrichment in coding regions and transcription start sites.

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 in silico analyses across 64 trypanosomatid species, uncovering a high degree of variability in G4 prevalence and species-specific patterns. We generated the first chromatin-based, genome-wide G4 map in T. brucei using G4 chromatin immunoprecipitation followed by sequencing (G4 ChIP-Seq), revealing enrichment within gene-associated regions, including coding DNA sequences (CDSs), and transcription boundaries such as transcription start sites (TSSs) and transcription termination sites. This pattern diverges markedly from previous genome-wide G4 maps in humans, suggesting that G4s may play roles unique to trypanosome biology. To investigate their functional relevance, we profiled the transcriptome of T. brucei upon treatment with the G4-stabilizing ligand PhenDC3. PhenDC3 induced targeted transcriptional perturbation of genes bearing G4s, particularly those located within CDSs and TSSs. Altogether, our findings highlight a distinctive role for G4s in regulating gene expression in T. brucei and support their potential as therapeutic targets in the treatment of African sleeping sickness.