Phase separation of ecDNA aggregates establishesin-transcontact domains boosting selectiveMYCregulatory interactions

Abstract

AbstractExtrachromosomal 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 andcis-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,MYC-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 establishin-transassociated contact domains (I-TADs) enriched, beyond copy number, in regulatory contacts among specific ecDNA regions, encompassing itsPVT1-MYCfusions but not its other canonicalMYCcopy. That explains why the fusions originate most of ecDNAMYCtranscripts (Hung et al., 2021), and shows that ecDNA clusteringper seis 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 pathogenicMYCupregulations. 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 reducesMYCtranscription (Hung et al., 2021).