Phase separation of ecDNA condensates establishes in-trans contact domains that boost selective MYC regulatory interactions.

Extrachromosomal DNAs (ecDNAs) are circular DNA elements found in many human cancers, often carrying oncogenes and regulatory elements. In COLO320-DM colorectal cancer cells, MYC-harboring ecDNAs form nuclear clusters associated with oncogene overexpression, but the mechanisms underlying their aggregation and gene upregulation remain unclear. Here, we use a minimal polymer physics model to investigate the interactions between ecDNA BRD4 binding sites and BRD4 molecules at the single-molecule level. The model demonstrates that BRD4 induces phase separation of ecDNAs condensates, forming in-trans associated contact domains (I-TADs) enriched, beyond copy number, in regulatory contacts among specific ecDNA regions, particularly the PVT1-MYC fusions. This explains the preferential transcriptional upregulation of these fusions, while the canonical copy of MYC on the ecDNAs remains unaffected. The formation of I-TADs is validated against independent HiChIP and ChIA-PET data. Regulatory contacts are strongly enriched as soon as half a dozen ecDNAs cluster, then saturate because of steric hindrance, highlighting that even cells with few ecDNAs can experience pathogenic MYC upregulations. The BET inhibitor JQ1 reverses ecDNA phase separation, abolishing I-TADs and reducing MYC transcription in a switch-like manner, as confirmed by RT-qPCR experiments. These findings clarify the role of ecDNA condensates in gene regulation and highlight potential therapeutic strategies targeting BRD4-mediated interactions.