Novel T. brucei genome assembly and high resolution 3D nuclear architecture

The nucleus of eukaryotic cells is highly organized, with RNA transcription and processing factors confined to specific nuclear structures. While most studies on 3D genome architecture have focused on intra-chromosomal interactions, such as promoter-enhancer dynamics and topologically associated domains, the significance of inter-chromosomal interactions remains less well understood. Studying inter-chromosomal DNA-DNA interactions in mammalian cells is challenging due to the large genome size, requiring deep sequencing. Additionally, complex transcription-dependent 3D topologies complicate the analysis of interaction patterns in mixed cell populations. To address these challenges, we used high-resolution DNA-DNA contact mapping (Micro-C) in Trypanosoma brucei. Here, RNA Pol II transcription is continuous, and parasites 9,000 genes are organized into polycistronic transcription units (PTUs), reducing the number of transcription start sites (TSSs) to approximately 200, greatly facilitating data interpretation. Additionally, to minimize artifacts from incorrectly scaffolded contigs, we generated ultra-long sequencing reads to close approximately 85% of gaps and collapsed repeat regions, resulting in a highly contiguous new genome assembly. Analyzing our Micro-C data with the new genome assembly revealed a nested 3D genome organization centered around polymerase-class-specific transcriptional hubs. Pol I-transcribed genes interact with each other as expected. However, we also found that Pol II TSSs form inter-chromosomal transcription hubs with other Pol II TSSs. Unlike the mouse or human genome, the T. brucei genome does not fold into distinct chromosome territories. Instead, chromosomes converge into numerous inter-chromosomal interaction hubs involving centromeres and polymerase-class-specific loci, highlighting the evolutionary significance of these structures.