Live-cell imaging shows uneven segregation of extrachromosomal DNA elements and transcriptionally active extrachromosomal DNA hubs in cancer.
Eunhee YiAmit D GujarMolly GuthrieHoon KimDacheng ZhaoKevin C JohnsonSamirkumar B AminMegan L CostaQianru YuSunit DasNathaniel JillettePatricia A ClowAlbert Wu ChengRoel G W VerhaakPublished in: Cancer discovery (2021)
Oncogenic extrachromosomal DNA elements (ecDNAs) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency greatly varies. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. EcTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. Post-mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis.
Keyphrases
- single cell
- copy number
- living cells
- induced apoptosis
- single molecule
- genome wide
- mitochondrial dna
- circulating tumor
- cell free
- squamous cell carcinoma
- transcription factor
- dna methylation
- type diabetes
- crispr cas
- cell therapy
- high resolution
- stem cells
- endoplasmic reticulum stress
- case report
- fluorescent probe
- photodynamic therapy
- high throughput
- circulating tumor cells
- squamous cell
- lymph node metastasis