First Genome-Wide Centromere Map of Trypanosoma cruzi Reveals Linear and 3D Compartment Boundaries and Spatial Clustering

Background: Trypanosoma cruzi , the etiological agent of Chagas disease, possesses a highly repetitive genome that has historically hindered high-quality assembly and structural characterization. Despite significant advances in assembling T. cruzi genomes, major gaps remain. Among these, the complete repertoire of centromeric sequences has remained elusive, representing a critical missing piece in our understanding of chromosome structure and inheritance . Results: Here, we generated high-coverage Hi-C (genome-wide chromosome conformation capture) data for the widely used T. cruzi Dm28c strain improving its genome assembly, reducing the number of scaffolds and producing a more contiguous and accurate genome. To investigate centromere organization, we performed ChIP-seq using the mNeonGreen-myc-tagged kinetochore proteins KKT2 and KKT3, resulting in the identification of 40 KKT-enriched peaks across 29 scaffolds. These peaks were located in regions enriched in retrotransposable elements, particularly L1Tc and VIPER, near strand switch regions, areas of high GC content, and at the boundaries between conserved genes and virulence-factor multigene families. Conclusion: Notably, Hi-C analysis revealed that centromeres may act as structural boundaries contributing to genome compartmentalization and frequently engage in 3D spatial clustering, suggesting a role in higher-order nuclear architecture. Overall, our study provides a high-quality reference genome for the Dm28c strain, presents the first genome-wide centromere map in T. cruzi , and offers novel insights into centromere-mediated 3D genome organization