Title

Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer.

Document Type

Article

Publication Date

2-2022

Publication Title

Cancer Discov

Keywords

JGM, DNA, Extrachromosomal Inheritance, Gene Amplification, Humans, Neoplasms, Tumor Microenvironment

JAX Source

Cancer Discov 2022 Feb; 12(2):468-483

Volume

12

Issue

2

First Page

468

Last Page

483

ISSN

2159-8290

PMID

34819316

DOI

https://doi.org/10.1158/2159-8290.cd-21-1376

Grant

CA237208, CA256575, CA236681, CA034196, HG00990

Abstract

Oncogenic extrachromosomal DNA elements (ecDNA) 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 varies greatly. 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. After 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. SIGNIFICANCE: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription.

Comments

We gratefully acknowledge the contribution of the Microscopy Service and the Single Cell Biology Laboratory at the Jackson Laboratory for expert assistance with the work described in this publication.

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