To characterise the role of DNA G-quadruplexes (G4s) in trypanosomes, we used G4-selective chromatin immunoprecipitation assays followed by high-throughput sequencing (G4 ChIP-seq, Figure 1A). ChIP-seq is typically used to determine the location of specific proteins within the entire genome of an organism. G4 ChIP-seq has been widely applied in human cells to develop a genomic map of G4s, however, so far there is no evidence of this technique performed in parasitic pathogens. Broadly, this complex technique required the expression of a G4-selective antibody using Escherichia coli cells, and preparation of parasite chromatin extracted from T. brucei cells cultured in vitro, which is segmented into DNA fragments of approx. 500 bp. Then, the optimal ChIP-seq assay conditions (e.g. antibody-to-chromatin ratio) were determined in order to obtain a successful enrichment of G4-structures in ChIP samples compared to the non G4-enriched controls (Input sample). Finally, a newly optimised bioinformatic pipeline was implemented to analyse the data derived from DNA sequencing. Results from preliminary ChIP-seq assays with T. brucei cells ultimately identified thousands of G4-forming sequences (i.e. G4-peaks) within the genome of the parasites. Specifically, 3,144 G4-peaks were identified to be shared between two experimental ChIP-replicates. The genomic distribution of the peaks shows a significant enrichment of G4s in exons (61%) and promoter regions (20%), compared to intergenic regions (5%) and transcription termination sites (TTS, 13%) (Figure 1B), suggesting an important biological role of G4s in transcriptional regulation. Notably, these results are in agreement with previously published bioinformatic predictions. Functional annotation of the G4-peaks revealed genes with important biological implications, including, for example, transcriptional regulation of the Variant Surface Glycoprotein (VSG). VSG represents the most important virulence factor in trypanosomes, helping the parasites to escape host immune responses and therefore causing prolonged infections. Two G4-peaks were identified in the bloodstream expression site (BES) 1, where VSG genes are actively transcribed. Interestingly, these G4-peaks are found in genomic regions nearby putative G4-sequences (PQS) identified by the bioinformatic G4-searching tool, G4Hunter (Figure 1C). These preliminary results highlight the potential role of G4s in controlling antigenic variation in trypanosomes and could be further exploited in the future to understand the link between DNA secondary structures and virulence control in infectious agents.
Work derived from this project was presented at national and international seminars and conferences with poster and oral presentations.
