Reconstructing the three-dimensional architecture of extrachromosomal DNA with ec3D.

Extrachromosomal DNAs (ecDNAs) are circular DNA molecules prevalent in human cancers that drive tumor evolution and drug resistance. Their circular topology, which disrupts topological domains and rewires regulatory circuits, has typically been studied via pairwise interactions. Here we develop ec3D, a computational method for reconstructing three-dimensional ecDNA structures from Hi-C data. Given a candidate ecDNA sequence and whole-genome Hi-C data, ec3D reconstructs spatial structures by maximizing the Poisson likelihood of observed interactions. We validate ec3D using simulated structures, previously characterized cancer cell lines, and microscopy imaging. Our reconstructions reveal that ecDNAs occupy spherical configurations and mediate unique long-range regulatory interactions involved in gene regulation. Through algorithmic innovations, ec3D can resolve complex structures with duplicated segments, identify multi-way interactions, and identify potential intermolecular (trans) interactions. Our findings provide insights into how ecDNA's spatial organization bypasses normal chromosomal constraints and contributes to increased oncogene expression.