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Transcripts derived from centromere repeats play a critical role in the localization and activity of kinetochore components during mitosis such that disruption of RNA polymerase II-dependent transcription compromises the fidelity of chromosome segregation. Here, we show that the retinoblastoma tumor suppressor protein (RB), a critical regulator of the G1/S cell cycle transition, additionally plays an important role in the regulation of centromere transcription during mitosis. We find that cells lacking RB experience increased RNA Polymerase II activity at mitotic centromeres and a corresponding increase in nascent RNA transcripts derived from centromere sequences. Together with high levels of centromere transcription and corresponding R-loop formation, RB-deficient cells exhibit centromere DNA breaks and local activation of ATR that correspond with increased centromere localization of Aurora B, destabilization of kinetochore-microtubule attachments, and an increase in anaphase defects. Importantly, reduction of DNA damage, ATR activity, and mitotic defects following inhibition of RNA Pol II, or targeted repression of centromere transcription through centromere tethering of Suv420h2, support that mitotic defects in RB-deficient cells are linked to centromere transcription.
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Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer
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. See related commentary by Henssen, p. 293. This article is highlighted in the In This Issue feature, p. 275
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- PAR ID:
- 10349580
- Date Published:
- Journal Name:
- Cancer Discovery
- Volume:
- 12
- Issue:
- 2
- ISSN:
- 2159-8274
- Page Range / eLocation ID:
- 468 to 483
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1158/2159-8290.CD-21-1376
