ecDNA hubs drive cooperative intermolecular oncogene expression [HiChIP]

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high oncogene expression through gene amplification and altered gene regulation. Gene induction typically involves cis regulatory elements that contact and activate genes on the same chromosome. Here we show that ecDNA hubs, clusters of ~10-100 ecDNAs within the nucleus, enable intermolecular enhancer-gene interactions to promote oncogene overexpression in trans. ecDNAs encoding multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumors. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the BET protein BRD4 in a MYC-amplified colorectal cancer cell line. BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-based oncogene transcription. A BRD4-bound promoter in PVT1 is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent MYC expression. PVT1 promoter on a heterologous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic CRISPRi silencing of ecDNA enhancers reveal intermolecular enhancer-gene activation among multiple oncogene loci amplified on distinct ecDNAs. Together, these results demonstrate that ecDNA hubs are protein-tethered clusters of ecDNAs which enable intermolecular transcriptional regulation. ecDNA hubs may act as units of oncogene function, cooperative evolution, and potential targets for cancer therapy. Overall design: H3K27ac HiChIP of ecDNA-containing cell lines COLO320-DM and SNU16.

染色体外DNA（extrachromosomal DNA, ecDNA）在人类癌症中广泛存在，并通过基因扩增与异常基因调控介导癌基因的高表达。基因诱导通常依赖顺式调控元件（cis regulatory elements），这类元件可在同一条染色体上接触并激活靶基因。本研究发现，ecDNA簇——即细胞核内约10~100个ecDNA聚集形成的动态簇状结构——可通过分子间增强子-基因反式（trans）相互作用，促进癌基因的过表达。
ecDNAs携带多种不同癌基因，可在多种癌细胞系及原发性肿瘤中形成簇状聚集体。当单个ecDNA与其他ecDNA在空间上相互聚集时，其携带的癌基因转录活性会显著增强。在MYC扩增的结直肠癌细胞系中，ecDNA簇由BET蛋白BRD4锚定。BET抑制剂JQ1可有效分散ecDNA簇，并优先抑制基于ecDNA的癌基因转录。PVT1基因座上一个结合BRD4的启动子发生异位融合至MYC并在ecDNA中发生扩增，可接收杂乱的增强子输入以驱动强效的MYC表达。将异源附加体上的PVT1启动子置入体系后，即可通过ecDNA簇以JQ1敏感的方式介导反式基因激活。对ecDNA增强子进行系统性CRISPR干扰（CRISPRi）沉默筛选发现，在不同ecDNA上扩增的多个癌基因位点之间存在分子间增强子-基因激活现象。
综上，本研究结果证实ecDNA簇是由蛋白锚定的ecDNA聚集结构，可介导分子间转录调控。ecDNA簇可作为癌基因功能、协同进化的功能单元，并有望成为癌症治疗的潜在靶点。
整体实验设计：对携带ecDNA的细胞系COLO320-DM与SNU16开展H3K27乙酰化修饰HiChIP检测。