Phase separation of ecDNA aggregates establishes contact domains boosting selective regulatory interactions.

Extrachromosomal DNAs (ecDNAs) are found in the nucleus of an array of human cancer cells where they can form clusters that were associated to oncogene overexpression, as they carry genes and -regulatory elements. Yet, the mechanisms of aggregation and gene amplification beyond copy-number effects remain mostly unclear. Here, we investigate, at the single molecule level, -harboring ecDNAs of COLO320-DM colorectal cancer cells by use of a minimal polymer model of the interactions of ecDNA BRD4 binding sites and BRD4 molecules. We find that BRD4 induces ecDNAs phase separation, resulting in the self-assembly of clusters whose predicted structure is validated against HiChIP data (Hung et al., 2021). Clusters establish associated contact domains (I-TADs) enriched, beyond copy number, in regulatory contacts among specific ecDNA regions, encompassing its fusions but not its other canonical copy. That explains why the fusions originate most of ecDNA transcripts (Hung et al., 2021), and shows that ecDNA clustering is important but not sufficient to amplify oncogene expression beyond copy-number, reconciling opposite views on the role of clusters (Hung et al., 2021; Zhu et al., 2021; Purshouse et al. 2022). Regulatory contacts become strongly enriched as soon as half a dozen ecDNAs aggregate, then saturate because of steric hindrance, highlighting that even cells with few ecDNAs can experience pathogenic upregulations. To help drug design and therapeutic applications, with the model we dissect the effects of JQ1, a BET inhibitor. We find that JQ1 reverses ecDNA phase separation hence abolishing I-TADs and extra regulatory contacts, explaining how in COLO320-DM cells it reduces transcription (Hung et al., 2021).