Hidden origami inTrypanosoma cruzinuclei highlights its nonrandom 3D genomic organization

Abstract

AbstractThe protozoanTrypanosoma cruzi, the causative agent of Chagas disease, exhibits polycistronic transcription and unidimensional genome compartmentalization ofcore(conserved) anddisruptive(virulence factors from multigenic families) genes. Approximately 50% of its genome is repetitive, mainly virulence factor genes. Genomic sequences, including repeats, motifs of architectural proteins, and noncoding RNA loci are crucial for genome folding. Here, we evaluated the genomic features associated with higher-order chromatin organization inT. cruzithrough extensive computational processing of high-throughput chromosome conformation capture (Hi-C) data, accounting for repetitive regions and improvements in genome annotation. Our study revealed that repetitive DNA (multimapped reads) influences 3D chromatin folding, particularly in determining the boundaries of topologically associated domains (TAD)-like structures. Virulence factor genes, unlikecoregenes, form shorter and more compact TAD-like structures enriched in loops, suggesting a gene expression regulatory mechanism. We found nonprotein-coding RNA loci (e.g., tRNAs) and transcription termination sites preferentially located at the boundaries of the TAD-like structures, while pseudogenes and multigenic family genes located in unstructured genomic regions. Our data indicate 3D clustering of tRNA loci, likely optimizing transcription by RNA polymerase III, and a complex interaction between spliced-leader RNA and 18S rRNA loci. Our findings provide insights into 3D genome organization inT. cruzi, contributing to the understanding of supranucleosome-level chromatin organization and suggesting possible links between 3D architecture and gene expression. We draw an analogy to the art of origami (e.g., papers folded into various shapes) resembling the DNA packed in chromatin fibers assuming distinct folds within the nucleus.ImportanceDespite the knowledge about the linear genome sequence and the identification of numerous virulence factors in the protozoan parasiteTrypanosoma cruzi, there has been a limited understanding of how these genomic features are spatially organized within the nucleus and how this organization impacts gene regulation and pathogenicity. By providing a detailed analysis of the three-dimensional chromatin architecture inT. cruzi, our study contributed to filling this gap. We deciphered part of the origami structure hidden in theT. cruzinucleus, showing the unidimensional genomic features are nonrandomly organized in the nuclear 3D landscape. We revealed the possible role of non-protein-coding RNA loci (e.g., tRNAs, SL-RNA, and 18S RNA) in shaping the genomic architecture. These findings provide insights into an additional epigenetic layer that may influence gene expression.Graphical abstractThe spatial organization of chromatin within the nuclei ofT. cruziand its resemblance to origami art. A. Identification of the 3D nuclear architectures withinT. cruzinuclei: topologically associating domains (TADs) and their boundaries; chromatin loops; and 3D networks. Inter- and intrachromosomal interactions reflect DNA‒DNA contacts on the same (cis) and between different (trans) chromosomes. B. Resemblance between origami art and chromatin folding. Steps “a” to “l” show the process of folding a flat piece of paper from its unidimensional view up to its 3D boat form.