Insight into the epigenetic regulation of gene expression in the bloodstream and procyclic forms of Trypanosoma brucei through the involvement of G-Quadruplexes
The study investigates the epigenetic role of G-quadruplexes (G4s) in the differentiation of Trypanosoma brucei brucei, specifically between its bloodstream form (BF) and procyclic form (PC). An in silico analysis identified 115,126 potential G4 sequences (PQSs) across the genome, with 63% having a high probability of G4 formation. These PQSs are unevenly distributed, with notable enrichment in regions associated with antigenic variation, such as variant surface glycoproteins and bloodstream expression sites. The differential distribution of PQSs correlates with regions of high densities of differentially expressed genes, suggesting a regulatory role in morphological transitions. The study assessed the effects of G4-ligands (AQ1, Pt-TTPY, and pyridostatin) on gene expression, revealing significant downregulation of DEGs and highlighting their therapeutic potential. Functional analysis using Gene Ontology indicated that G4-ligands impact various biological processes differently in BF and PC forms. These findings underscore the epigenetic complexity of T. brucei and the potential of G4 structures as key regulators of gene expression and differentiation, offering novel insights into therapeutic strategies targeting the parasite's adaptive mechanisms. Overall design: T. brucei parasites in logarithmic phase were incubated with 2X of the half-maximal inhibitory concentration of AQ1 (BSF: 0,24 uM and PCF: 9), Pt-TTPY (BSF: 0,26 uM and PCF: 48,46 uM) and pyridostatin (BSF: 11,04 uM and PCF: 18,18 uM) for 18 h at 37ºC or 28 ºC,  5% CO2 and 100% of humidity. Then, the parasites were washed twice in phosphate buffered saline and processed by RNA isolation. Total RNA was purified using a total RNA isolation kit (Qiagen, Germany) according to the manufacturer's instructions. The RiboCop for Bacteria (Lexogen) was used to deplete 800 ng of the extracted total RNA for rRNA. The NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, UK) was used to prepare the library preps for Illumina sequencing. Samples were sequenced on the Illumina NexSeq 1000 to generate 100 bases single-end reads (100SE) with an average read-depth of 10M reads per sample. The quality of the resulting fastq reads were checked using FastQC v0.11.9 (Babraham Bioinformatics, Cambridge) and mapped on the reference genome using STAR v2.7.10b (default setting with following changes; --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNoverReadLmax 0.04). The resulting SAM files were converted to BAM using SAMtools 1.1171 , and featuresCounts 2.0.1 72 was used to obtain the gene counts. Each sample was performed in duplicate